Compositions, methods, and kits for eliciting an immune response

ABSTRACT

The present invention relates to compositions, methods, and kits for eliciting an immune response to at least one CMV antigen expressed by a cancer cell, in particular for treating and preventing cancer. CMV determination methods, compositions, and kits also are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC §119 to U.S. ProvisionalApplication No. 61/074,582, filed Jun. 20, 2008, which is hereinincorporated by reference in its entirety.

GOVERNMENT INTERESTS

The United States Government has certain interests in this applicationvia grants from the National Institutes of Health, National CancerInstitute, and National Institute of Neurological Disorders and Stroke(NINDS SPORE in Brain Cancer P50-CA1-0876; NCI Dendritic CellImmunotherapy of Malignant Glioma R01 CA 97222).

FIELD OF THE INVENTION

The present invention relates to compositions, methods, and kits foreliciting an immune response to a cell that expresses a cytomegalovirus(CMV) antigen. The present invention also relates to methods,compositions, and kits for determining CMV.

BACKGROUND OF THE INVENTION

Methods for treating cancers include the use of chemotherapeutics,radiation therapy, and surgery. The identification of a number of tumorantigens has led to attempts at developing cell-based therapies. Somemethods have relied on first identifying a tumor antigen, i.e., apolypeptide that is expressed preferentially in tumor cells, relative tonon-tumor cells. For example, several human tumor antigens have beenisolated from melanoma patients, and identified and characterized.

CMV is a β-herpesvirus. Human cytomegalovirus (HCMV) is endemic in thehuman population and it has been reported that the virus does notusually cause clinical disease except in immunocompromised hosts. Somehuman herpesviruses have been implicated in a number of humanmalignancies including lymphoma, nasopharyngeal cancer, cervical cancer,and Kaposi's sarcoma. Recently, HCMV antigen expression and detection ofintact virus has been reported to occur in some tumors.

Despite aggressive multi-modality therapy including surgery, radiation,and chemotherapy, the prognosis for patients with cancer remainsrelatively poor. Moreover, the non-specific nature of conventionaltherapy for cancer often results in incapacitating damage to surroundingnormal and systemic tissues. Thus, there is a need for the developmentof effective diagnostic as well as therapeutic and prophylacticstrategies that target cancer cells.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of eliciting in asubject an immune response to a cell that expresses a cytomegalovirus(CMV) antigen. The method comprises: administering to the subject apharmaceutically acceptable composition comprising at least one CMVantigen, or nucleic acids encoding the at least one CMV antigen, whereinthe pharmaceutically acceptable composition, when administered to thesubject, elicits an immune response to the cell.

In another aspect, the present invention provides a pharmaceuticallyacceptable composition comprising at least one CMV antigen, or nucleicacids encoding the at least one CMV antigen, wherein thepharmaceutically acceptable composition, when administered to thesubject, elicits an immune response to a cell that expresses a CMVantigen.

In some aspects, a prophylactically or therapeutically effective amountof a pharmaceutically acceptable composition is provided by the presentinvention, wherein the pharmaceutically acceptable composition comprisesat least one CMV antigen, or nucleic acids encoding the at least one CMVantigen, wherein the pharmaceutically acceptable composition, whenadministered to the subject, elicits an immune response to a cell thatexpresses a CMV antigen.

In other aspects, the present invention provides a method of elicitingin a subject an immune response to a cell that expresses a CMV antigen,the method comprising:

administering to the subject a composition comprising an effectiveamount of antigen presenting cells, T-lymphocytes, or both, wherein theantigen presenting cells and T lymphocytes have been sensitized in vitrowith a sensitizing-effective amount of at least one CMV antigen, whereinthe effective amount of antigen presenting cells, T lymphocytes, or bothis sufficient to elicit the immune response to the cell that expressesthe CMV antigen.

In one aspect, the present invention provides a method for making anantigen-presenting cells, the method comprising: contactingantigen-presenting cells with at least one CMV antigen, or nucleic acidsencoding the at least one CMV antigen, in vitro under a conditionsufficient for the at least one CMV antigen to be presented by theantigen-presenting cells, wherein the antigen-presenting cell presentsthe at least one CMV antigen.

In still a further aspect, the present invention provides a compositioncomprising antigen-presenting cells contacted with at least one CMVantigen, or nucleic acids encoding the at least one CMV antigen, invitro under a condition sufficient for the at least one CMV antigen tobe presented by the antigen-presenting cells.

In some aspects, the present invention provides a method for makinglymphocytes, the method comprising:

a) contacting antigen-presenting cells with at least one CMV antigen, ornucleic acids encoding the at least one CMV antigen, in vitro under acondition sufficient for the at least one CMV antigen to be presented bythe antigen-presenting cells; and

b) contacting lymphocytes with the antigen-presenting cells of step a)under conditions sufficient to produce the lymphocytes, wherein thelymphocytes are capable of eliciting an immune response against a cellthat expresses a CMV antigen.

In other aspects, the present invention provides a compositioncomprising T lymphocytes contacted with antigen-presenting cells underconditions sufficient to produce T lymphocytes capable of eliciting animmune response against a cell that expresses a CMV antigen, wherein theantigen-presenting cells have been contacted with at least one CMVantigen, or nucleic acids encoding the at least one CMV antigen, invitro under a condition sufficient for the at least one CMV antigen tobe presented by the antigen-presenting cells.

In one aspect, a method for treating or reducing the severity of cancerin a subject is provided by the present invention. The method comprises:administering to the subject a therapeutically or prophylacticallyeffective amount of a composition comprising T lymphocytes contactedwith antigen-presenting cells under conditions sufficient to produce Tlymphocytes capable of eliciting an immune response against a cell thatexpresses a CMV antigen, wherein the antigen-presenting cells have beencontacted with at least one CMV antigen, or nucleic acids encoding theat least one CMV antigen, in vitro under a condition sufficient for theat least one CMV antigen to be presented by the antigen-presentingcells.

In another aspect, the present invention provides a method for elicitingin a subject an immune response to a cell that expresses a CMV antigen.The method comprises:

administering to the subject a pharmaceutically acceptable compositioncomprising dendritic cells loaded ex vivo with at least one CMV antigen,or nucleic acids encoding the at least one CMV antigen, wherein thepharmaceutically acceptable composition, when administered to thesubject, elicits an immune response to the cell that expresses a CMVantigen.

In some aspects, the present invention provides a method of treating acell that expresses a CMV antigen, the method comprising administeringto a subject a therapeutically or prophylactically effective amount of apharmaceutically acceptable composition to reduce or inhibit growth orspread of the cell in the subject, wherein the composition comprises:

a) at least one CMV antigen or a polynucleotide encoding the at leastone CMV antigen;

b) an anti-CMV antibody;

c) an antigen-presenting cell presenting the at least one CMV antigen, alymphocyte primed against the CMV antigen, or both; or

d) a combination thereof.

In other aspects, the present invention provides a method of elicitingin a subject an immune response to a cell that expresses a CMV antigen,the method comprising:

administering to the subject a composition comprising an effectiveamount of antigen-presenting cells, lymphocytes, or both, wherein theantigen-presenting cells and lymphocytes have been sensitized in vitrowith a sensitizing-effective amount of at least one CMV antigen, whereinthe effective amount of antigen-presenting cells, lymphocytes, or bothis sufficient to elicit the immune response to the cell that expressesthe CMV antigen.

In one aspect, the present invention provides a method of treating acell that expresses a CMV antigen, the method comprising administeringto a subject a composition comprising an effective amount ofantigen-presenting cells, lymphocytes, or both, wherein theantigen-presenting cells have been in vitro contacted with at least oneCMV antigen, or nucleic acids encoding the at least one CMV antigen,under a condition sufficient for the at least one CMV antigen to bepresented by the antigen-presenting cells, wherein the lymphocytes havebeen contacted with antigen-presenting cells presenting the at least oneCMV antigen.

In another aspect, the present invention provides a method of elicitingin a subject an immune response to a cell that expresses a CMV antigen,the method comprising:

administering to the subject a pharmaceutically acceptable compositioncomprising an anti-CMV antibody.

In various other aspects, the present invention provides compositionsand methods for determining CMV nucleic acid in a subject, preferablyCMV DNA in blood or other biological fluid, for example determiningsubclinical viremia in a sample of blood obtained from the subject.Accordingly, the compositions and methods provide diagnostic,monitoring, and prognostic tests/assays that complement variousdiagnostic and/or therapeutic procedures and treatments includingmethods described herein such as, for example, prophylactic and/ortherapeutic treating of a disease or condition associated with aprecancerous cell, a cancer cell, or a cell-type predisposed todeveloping cancer associated with CMV.

In other aspects, the present invention provides a kit comprising apharmaceutically acceptable composition comprising at least one CMVantigen, or nucleic acids encoding the at least one CMV antigen, whereinthe pharmaceutically acceptable composition, when administered to asubject, elicits an immune response against a cell that expresses a CMVantigen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows: (A) Immunohistochemical detection of human cytomegalovirus(HCMV) proteins: (a) negative control (no primary antibody; objectivelens ×10); (b) antismooth-muscle actin (mouse IgG2a mAb; ×10); (c)glioblastoma multiforme (GBM) specimen 1 stained with anti-HCMV IE1(mouse IgG2a mAb, ×10); (d) higher magnification of anti-IE1 stainingshows positive tumor cells and endothelial cells but negativelymphocytes and vascular intima (×20); (e) GBM specimen 2 stained withanti-HCMV IE1 showing staining of perivascular tumor cells but lack ofdetection in necrotic areas (×10); (0 perivascular tumor cells stainedwith anti-IE1 mAb (×20); (g and h) GBM specimen 3 stained with anti-HCMVpp65 mAb showing nuclear and perinuclear staining of tumor cellsscattered throughout the GBM specimen (×10 and ×20, respectively); (iand j) CMV-infected lung stained with antismooth-muscle actin mAb (×10);(k) CMV-infected lung stained with anti-HCMV IE1 (×20); (l) CMV-infectedlung stained with anti-HCMV pp65 mAb (×20); and (B) HCMV detection inmatched GBM and normal brain. Representative histochemical sections fromtwo GBM specimens containing areas of normal brain and tumor werestained for detection using isotype control antibodies (patient 1, leftcolumn), or anti-HCMV pp65 (patient 1 tumor, middle column; patient 2,right column). Focal areas of reactivity against the HCMV pp65 antibodywas observed throughout the tumor-involved areas, but normal brain wasdevoid of immunoreactivity to the HCMV-specific antibodies (IE1 stainingshowed identical findings with more ubiquitous detection of IE1 in thetumor, not shown). All photographs taken at ×40 objective magnification.

FIG. 2 shows polymerase chain reaction (PCR) detection of HCMV inmalignant glioma specimens. Lane 1: 100 bp ladder, Lanes 2-E: MGspecimens; Lane 4: MG sample 4+100 bp ladder; Lane F: negative control(no DNA template).

FIG. 3 shows a schematic depiction of a vaccination protocol forsubjects with newly-diagnosed GBMs using CMV pp65-LAMP RNA-loaded DCsduring recovery from therapeutic TMZ-induced lymphopenia with or withoutautologous Lymphocyte transfer (ALT) in subjects that are seropositiveand seronegative for CMV.

FIG. 4 is a graph showing time to progression for patients withnewly-diagnosed GBM treated with CMV pp65 RNA loaded DCs. Thirteenpatients with GBM were treated with DC vaccines targeting CMV pp65. Timeto progression is favorable compared to historical controls (P=0.0008).

FIG. 5 is a graph showing overall survival of patients with GBM treatedwith CMV pp65 RNA loaded DC vaccines.

FIG. 6 is a magnetic resonance image (MRI) showing near completeradiographic response during pp65 RNA loaded DC vaccination for a 33year-old female subject with GBM.

FIG. 7 shows analysis of polyfunctional T cell responses to CMV pp65.Peripheral blood mononuclear cells (PBMCs) from a CMV seropositive donorwere stimulated in vitro with CMV pp65 peptide mix and analyzed using avalidated 11-color polychromatic flow cytometric analysis in theImmunologic Monitoring Core laboratory of the HIV Vaccine TrialsNetwork. Functional and Boolean gating software was used to determinethe proportion of polyfunctional T cells (secreting multiple cytokinesand displaying granzyme activation marker CD107a). Polyfunctional immuneresponses have been shown to distinguish progressors versusnon-progressors with HIV infection and have correlated better withimmune protection in viral infection than absolute numbers orpercentages of cells secreting a particular cytokine.

FIG. 8 is a graph showing increase in pp65 antibody response in apatient with newly-diagnosed GBM receiving vaccination with autologousdendritic cells pulsed with pp65 mRNA. The panel on the left showsstandard curve binding of a pp65-specific monoclonal antibody to beadscoated with recombinant pp65 protein. The right panel shows specificbinding of antibodies present in dilutions of a patient's serum to beadscoated with recombinant pp65. Pre-vaccine serum contains low titer ofpp65-specific antibody and increasing titer (shown as mean fold increase(MFI) after detection with anti-human IgG fluorescent antibodies) atvaccine #5 and further increase by vaccine #9. Furthermore, theseresponses were induced during cycles of temozolomide, demonstrating thecapacity to induce potent humoral responses during concurrentchemotherapy administration.

FIG. 9 is a bar graph showing increase in delayed-type hypersensitivity(DTH) responses in patients with newly-diagnosed GBM receivingvaccination with autologous dendritic cells pulsed with pp65 mRNA. Theproportion of patients demonstrating a positive DTH response (≧10 mm²)prior to and after vaccination to pp65 and control antigens (tetanus andCandida) is shown on the left; and area of skin erythema prior to andafter vaccination is shown on the right. Furthermore, these responseswere induced during cycles of temozolomide, demonstrating the capacityto induce cellular DTH responses during concurrent chemotherapyadministration.

FIG. 10 is a fluorescence-activated cell sorting (FACS) profile.Nonadherent cells from PBMCs were stimulated in vitro, usinganti-CD3-coated plates, for 72 hr and harvested, washed, andelectroporated with RNA encoding green fluorescent protein (GFP) or CXCchemokine receptor 2 (CXCR2) (2 μg of RNA per 10⁶ cells) as described inExamples 10 & 11 below. Cells were cultured in medium (AIM-V medium, 2%AB serum, hIL-2 [100 U/ml]) for 48 hr, washed, and analyzed for GFPexpression and CXCR2 by administration of allophycocyanin(APC)-conjugated CXCR2-specific monoclonal antibody and flow cytometry.Top: Untransfected T cell expression of GFP (top left) and CXCR2 (topright). Bottom: GFP expression after electroporation with GFP RNA(bottom left) and CXCR2 expression after electroporation with CXCR2 RNA(bottom right). This experiment was repeated twice with similar results.

FIG. 11 is a FACS profile. PBMCs from an HLA-A2-positive donor werestimulated for 7 days with autologous DCs pulsed with anHLA-A2-restricted pp65 peptide (N9V), harvested, and electroporated withGFP RNA. Forty-eight hours later GFP expression was examined inN9V-tetramer-positive versus N9Vtetramer-negative CD8+ T cells. Left:Gating on blastlike lymphocytes (R1; top) and CD8+ T cells (R2; bottom).Middle: No GFP expression in cells transfected with a control RNA(CXCR2). Right: GFP expression in 24.57% of CD8+ T cells (top right),which consists almost entirely of pp65-specific T cells as shown bytetramer staining (gated on lymphocytes (R1) and CD8+ cells (R2))(bottom right).

FIG. 12 is a graph showing percentage of GFP-expressing cells and FACSprofiles. (A) Kinetics of GFP expression in RNA-transfected T cellsstimulated with DCs. PBMCs were stimulated for 11 days in vitro withautologous DCs pulsed with pp65 mRNA as described in Examples 10 & 11below. Cells were harvested and transfected with GFP RNA (2 μg/106cells). Expression of GFP in tetramer-positive (HLA-A2B) andtetramer-negative cells was evaluated beginning 24 hr afterelectroporation and monitored until day 7 postelectroporation. (B)Expansion of sorted GFP RNA-transfected T cells. PBMCs were stimulatedfor 11 days in vitro with autologous DCs pulsed with pp65 mRNA asdescribed in Materials and Methods. Cells were harvested and transfectedwith GFP RNA (2 μg/10⁶ cells). Forty-eight hours later, cells weresorted by flow cytometry on the basis of GFP expression (GFP+ and GFP−)and placed back into culture with high-dose IL-2. Expansion of the cellswas evaluated by trypan blue staining and counting of an aliquot ofcells every 3-4 days. This experiment was repeated with PBMCs fromanother donor with similar results. (C) Tetramer analysis of HLA-A2- andHLA-B7-restricted pp65-specific T cells. A panel of tetramers was usedto identify patients with more than one type of haplotype-restricted Tcell reactivity against pp65 detectable by tetramer. Left: Frequency ofHLA-A2- and HLA-B7 pp65-specific T cells in the GFP− fraction. Right:Frequency of tetramer-positive cells in the GFP fraction. (D) Isolationof CMV antigen-specific CD4 T cells by RNA electroporation. PBMCsstimulated with pp65 RNA pulsed DCs were generated as previouslydescribed and electroporated with GFP RNA on day 11 after DCstimulation. Cells were sorted into GFP+ and GFP− fractions and 48 hrlater were analyzed by intracellular cytokine flow cytometry for IFN-Bproduction after exposure to no antigen, SEB (1 Bg/ml), or CMV pp65peptide mix (Beckman Coulter). Left: Expression of IFN-B in GFP− CD4+ Tcells. Right: IFN-B in GFP+ CD4+ T cells.

FIG. 13 is a FACS profile. Selective expression of CXCR2 inpp65-specific T cells after RNA electroporation. PBMCs were stimulatedfor 11 days in vitro with autologous DCs pulsed with pp65 mRNA asdescribed in Examples 10 & 11. Cells were harvested and transfected withCXCR2 RNA (2 μg/10⁶ cells). Forty-eight hours later, cells wereharvested and analyzed for CXCR2 expression in tetramer-positive(HLA-A2-restricted donor) and tetramer-negative CD8+ cells. Baselineexpression in activated CD8+ T cells of CXCR2 was approximately 10%(data not shown). Results demonstrated that RNA electroporation resultedin increased CXCR2 expression in CMV-specific T cells only, withexpression in 49.1% of tetramer-positive cells, whereas no increase inexpression over baseline was observed in tetramer-negative cells afterDC stimulation.

FIG. 14 is a graph. (A) In vitro chemotaxis of CXCR2 and GFPRNA-transfected T cells toward IL-8. PBMCs were activated in vitro usingimmobilized anti-CD3 antibody-mediated stimulation for 4 days asdescribed in Examples 10 & 11. Cells were harvested and transfected withCXCR2 or GFP RNA (2 μg/10⁶ cells). Forty eight hours later, cells wereharvested, counted, and placed in triplicate into filter chamber cultureplates as described in Examples 10 & 11. The migration of untransfected,GFP RNA transfected, and CXCR2 RNA-transfected T cells in response toincreasing concentrations of IL-8 was assessed after 45 min of culturein the presence of the indicated concentration of IL-8 in the lowerchamber. Results demonstrated enhanced chemotactic response of CXCR2 RNAtransfected T cells compared with untransfected and GFP RNA-transfectedcells (*p<0.05; t test). This experiment has been repeated several timeswith the same results. (B) In vitro chemotaxis of CXCR2 and GFPRNA-transfected T cells toward GRO-α. Cells were assayed as describedabove against increasing concentrations of GRO-α in the lower chamber ofthe filter chamber plates. CXCR2-transfected T cells exhibitedsignificantly increased chemotactic activity in response to GRO-α,whereas GFP RNA-transfected and untransfected T cells showed nosignificant change in migration in response to GRO-α (*p<0.05; t test).(C) In vitro chemotaxis of CXCR2 and GFP RNA transfected T cells towardUL146. Cells were assayed as described above against increasingconcentrations of the CMV-secreted chemokine UL146 in the lower chamberof the filter chamber plates. CXCR2-transfected T cells exhibitedmarkedly increased chemotactic activity in response to UL146, whereasGFP RNA-transfected and untransfected T cells showed no change inmigration in response to UL146 (**p<0.01; t test).

FIG. 15 is a graph and FACS profile. (A) Migration of CXCR2RNA-transfected T cells into the peritoneal cavity. PBMCs werestimulated for 11 days in vitro with autologous DCs pulsed with pp65mRNA as described in Examples 10 & 11 below. Cells were harvested andtransfected with CXCR2 RNA (2 Bg/106 cells). Forty-eight hours later,untransfected and CXCR2 RNA-transfected cells were harvested anddifferentially labeled with CFSE (1 μM for untransfected cells and 10 BMfor CXCR2 RNA-transfected cells) for 5 min in PBS, washed, mixed, andinjected intravenously into SCID mice (2×10⁷ cells per mouse, n=3 pergroup). Mice were injected intraperitoneally with PBS only or withchemokines (GRO-α, IL-8, or UL146) at 1 μg/ml in PBS every 8 hr for 24hr after injection of T cells. Lymphocytes were harvested aftersacrifice by peritoneal lavage and centrifuged, and the relativeaccumulation of untransfected and CXCR2 RNA-transfected cells wasevaluated by flow cytometric analysis. Accumulation of CFSE^(high) cells(CXC2 RNA transfected T cells) and CFSE^(low) cells (untransfected Tcells) in PBS-treated animals was used as a baseline for comparison withthe accumulation of cells in cytokine-treated animals. Data aredisplayed as fold change compared with PBS-treated animals. (B)Migration of CXCR2 RNA-transfected T cells into the CNS. PBMCs werestimulated for 11 days in vitro with autologous DCs pulsed with pp65mRNA as described in Examples 10 & 11. Cells were harvested andtransfected with CXCR2 RNA (2 μg/10⁶ cells). Forty-eight hours later,untransfected and CXCR2 RNA-transfected cells were harvested and labeledwith CFSE (5 μM) for 5 min in PBS, washed, and injected intravenouslyinto SCID mice (1×10⁷ cells per mouse, n=3 per group). Anesthetized micewere subsequently injected via the right parietal lobe with the CMVchemokine UL146 (1 μg in 5 μl of PBS) and via the left parietal lobewith PBS only. The accumulation of CFSE-labeled cells in the right andleft parietal lobes was assessed 6 hr later by flow cytometry afterdissection of the cerebral hemispheres and single-cell digestpreparation from the parietal lobes. A representative flow cytometricdot plot of a mouse injected with CXCR2 RNA-transfected T cells showsgreater accumulation of lymphocytes within the right parietal lobeinjected with the CXCR2 ligand UL146. (C) In vivo migration ofCXCR2-transfected T cells. The accumulation of T cells within the right(UL146-injected) and left (PBS-injected) parietal lobes was assessed byflow cytometry and human T cells per 100,000 events were plotted.Results demonstrated significant accumulation of CXCR2 RNA-transfected Tcells within the CNS in response to UL146, whereas untransfected T cellsdid not exhibit any preferential accumulation (*p<0.05; t test).

FIG. 16 is an electrophoretic gel showing amplification of CMV gB (UL55)DNA from peripheral blood and tumors of patients with GBM. A: Lane 1:100 bp DNA ladder; Lanes 2-12: Peripheral blood samples from 11 patientswith newly-diagnosed GBM taken at time of primary resection of tumorshows strong detection of viral DNA in 6 out of 11 patients and a weakband of appropriate size in 3 out of 11 patients. B: Lane 1: 100 bp DNAladder; Lanes 2-F: GBM tumor samples from 14 patients with high gradeastrocytomas (11 GBM, 3 AA) shows clear band of appropriate size in 11out of 14 patients. Lane 4 has 100 bp DNA ladder mixed with sample foralignment with amplified bands.

FIG. 17 is a melting curve graph of HCMV gp64 Real-Time PCR from GBMpatient sera.

FIG. 18 is a PCR amplification cycle graph of HCMV gp64 Real-Time PCRfrom GBM patient sera.

FIG. 19 shows sequence homology. The amplified gB (UL55) DNA from 22freshly resected GBM specimens was evaluated by DNA sequencing performedin the Duke DNA Sequencing Core Facility. Sequence alignment wasperformed using Vector NTI Advance 10 (Invitrogen, Carlsbad, Calif.).Homology to identified CMV strains was evaluated by BLAST of NCBI DNAdatabase. Areas of sequence variance between samples are shown in blackwith nucleotide differences highlighted in white. Dotted indicatesconserved identity throughout all 22 specimens. White and blackindicates areas of sequence variation.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have discovered that cells that express a CMV antigen can betargeted using CMV-specific immunological techniques includingimmunotherapy involving, for example, vaccines. The invention isapplicable, but not limited, to the development of compositions,methods, and kits for diagnostics and therapies for cells that express aCMV antigen.

I. Definitions

The term “immune response” refers herein to any response to an antigenor antigenic determinant by the immune system. Exemplary immuneresponses include humoral immune responses (e.g. production ofantigen-specific antibodies (neutralizing or otherwise)) andcell-mediated immune responses (e.g. lymphocyte proliferation).

II. Methods and Compositions

In one aspect, the present invention provides a method of eliciting in asubject an immune response to a cell that expresses a CMV antigen. Themethod comprises: administering to the subject a pharmaceuticallyacceptable composition comprising at least one CMV antigen, or nucleicacids encoding the at least one CMV antigen, wherein thepharmaceutically acceptable composition, when administered to thesubject, elicits an immune response to the cell.

The cell that expresses a CMV antigen can be any type of cell. The cellcan be characterized as a cancer cell, a precancerous cell, or acell-type predisposed to developing cancer associated with CMV. Thecancer includes but is not limited to brain cancers (e.g., gliomas),lung cancers, liver cancers, cervical cancers, soft tissue sarcomas,endocrine tumors, hematopoietic cancers, melanomas, bladder cancers,breast cancers, pancreatic cancers, prostate cancers, colon cancers, andovarian cancers. The cancer also can be characterized as benign ormalignant. In one embodiment, the cancer is a high grade glioma. Inanother embodiment, the high grade glioma is a glioblastoma multiforme,an anaplastic astrocytoma, or an oligodendroglioma.

Generally, the immune response can include the humoral immune response,the cell-mediated immune response, or both. For example, antigenpresentation through an immunological pathway involving MHC II proteinsor direct B-cell stimulation can produce a humoral response; and,antigens presented through a pathway involving MHC I proteins can elicitthe cellular arm of the immune system.

A humoral response can be determined by a standard immunoassay forantibody levels in a serum sample from the subject receiving thepharmaceutically acceptable composition. A cellular immune response is aresponse that involves T cells and can be determined in vitro or invivo. For example, a general cellular immune response can be determinedas the T cell proliferative activity in cells (e.g., peripheral bloodleukocytes (PBLs)) sampled from the subject at a suitable time followingthe administering of a pharmaceutically acceptable composition.Following incubation of e.g., PBMCs with a stimulator for an appropriateperiod, [³H]thymidine incorporation can be determined. The subset of Tcells that is proliferating can be determined using flow cytometry. Tcell cytotoxicity (CTh) can also be determined.

In one embodiment, the immune response that is elicited is sufficientfor prophylactic or therapeutic treatment of a neoplastic disease, or asymptom associated therewith, particularly cancer associated with CMV.Accordingly, a beneficial effect of the pharmaceutically acceptablecomposition will generally at least in part be immune-mediated, althoughan immune response need not be positively demonstrated in order for thecompositions and methods described herein to fall within the scope ofthe present invention.

Administering to both human and non-human vertebrates is contemplatedwithin the scope of the present invention. Veterinary applications alsoare contemplated. Generally, the subject is any living organism in whichan immune response can be elicited. Examples of subjects include,without limitation, humans, livestock, dogs, cats, mice, rats, andtransgenic species thereof.

The subject can either have a neoplastic disease (e.g., a tumor), or beat risk of developing the neoplastic disease. Subjects can becharacterized by clinical criteria, for example, those with advancedneoplastic disease or high tumor burden exhibiting a clinicallymeasurable tumor. A clinically measurable tumor is one that can bedetected on the basis of tumor mass (e.g., by palpation, MRI, CAT scan,X-ray). Thus, for example, the pharmaceutically acceptable compositionin accordance with the present invention can be administered to subjectswith advanced disease with the objective of mitigating their condition.Preferably, a reduction in tumor mass occurs as a result ofadministering the pharmaceutically acceptable composition of the presentinvention, but any clinical improvement constitutes a benefit. Clinicalimprovement includes decreased risk or rate of progression or reductionin pathological consequences of a tumor, for example.

By way of another example, the subject can be one that has a history ofcancer and has been responsive to another mode of therapy. The othertherapy may have included e.g., surgical resection, radiotherapy,chemotherapy, and other modes of immunotherapy whereby as a result ofthe other therapy, the subject presents no clinically measurable tumor.However, the subject can be one determined to be at risk for recurrenceor progression of the cancer, either near the original tumor site, or bymetastases. Such subjects can be further categorized as high-risk andlow-risk subjects. The subdivision can be made on the basis of featuresobserved before or after the initial treatment. These features are knownin the clinical arts, and are suitably defined for each differentcancer. Features typical of high risk subgroups are those in which thetumor has invaded neighboring tissues, or which show involvement oflymph nodes. Thus, for example, a pharmaceutically acceptablecomposition of the present invention can be administered to the subjectto elicit an anti-cancer response primarily as a prophylactic measureagainst recurrence. Preferably, administering the composition delaysrecurrence of the cancer, or more preferably, reduces the risk ofrecurrence (i.e., improves the cure rate). Such parameters can bedetermined in comparison with other patient populations and other modesof therapy.

The pharmaceutically acceptable composition can be administered at anytime that is appropriate. For example, the administering can beconducted before or during traditional therapy of a subject having atumor burden, and continued after the tumor becomes clinicallyundetectable. The administering also can be continued in a subjectshowing signs of recurrence.

The pharmaceutically acceptable composition can be administered in atherapeutically or a prophylactically effective amount, wherein thepharmaceutically acceptable composition comprises the at least one CMVantigen, or nucleic acids encoding the at least one CMV antigen, eitheralone or in combination with one or more other antigens. Administeringthe pharmaceutically acceptable composition of the present invention tothe subject can be carried out using known procedures, and at dosagesand for periods of time sufficient to achieve a desired effect. Forexample, a therapeutically or prophylactically effective amount of thepharmaceutically acceptable composition, can vary according to factorssuch as the age, sex, and weight of the subject. Dosage regima can beadjusted by one of ordinary skill in the art to elicit the desiredimmune response including immune responses that provide therapeutic orprophylactic effects.

The pharmaceutically acceptable composition can be administered to thesubject at any suitable site, for example a site that is distal to orproximal to a primary tumor. The route of administering can beparenteral, intramuscular, subcutaneous, intradermal, intraperitoneal,intranasal, intravenous (including via an indwelling catheter), via anafferent lymph vessel, or by any other route suitable in view of theneoplastic disease being treated and the subject's condition.Preferably, the dose will be administered in an amount and for a periodof time effective in bringing about a desired response, be it elicitingthe immune response or the prophylactic or therapeutic treatment of theneoplastic disease and/or symptoms associated therewith.

The pharmaceutically acceptable composition can be given subsequent to,preceding, or contemporaneously with other therapies including therapiesthat also elicit an immune response in the subject. For example, thesubject may previously or concurrently be treated by chemotherapy (e.g.,by an alkylating agent such as temozolomide), radiation therapy, andother forms of immunotherapy, such other therapies preferably providedin such a way so as not to interfere with the immunogenicity of thecompositions of the present invention.

Administering can be properly timed by the care giver (e.g., physician,veterinarian), and can depend on the clinical condition of the subject,the objectives of administering, and/or other therapies also beingcontemplated or administered. In some embodiments, an initial dose canbe administered, and the subject monitored for either an immunologicalor clinical response, preferably both. Suitable means of immunologicalmonitoring include using patient's peripheral blood lymphocyte (PBL) asresponders and neoplastic cells as stimulators. An immunologicalreaction also can be determined by a delayed inflammatory response atthe site of administering. One or more doses subsequent to the initialdose can be given as appropriate, typically on a monthly, semimonthly,or preferably a weekly basis, until the desired effect is achieved.Thereafter, additional booster or maintenance doses can be given asrequired, particularly when the immunological or clinical benefitappears to subside.

A. CMV Antigens

In one embodiment, the at least one CMV antigen is a polypeptide, or animmunogenic fragment thereof, encoded by a CMV gene. As indicated above,the term “CMV,” as used herein, includes any strain of the virus thatinfects an animal, for example mammals such as humans and monkeys.Strains of CMV that infect humans are typically designated as human CMV(HCMV). ORFs and/or their corresponding polypeptides from HCMV can bereferred to using nomenclature as described by, for example, Chee etal., Curr. Top. Microbiol. Immunol., 154:125 (1990) and Spaete et al.,J. General Virology, 74:3287 (1994), each of which is incorporatedherein by reference for their teaching of such polypeptides and theirnomenclature. Reference to such reading frames and polypeptides from CMValso can refer to corresponding sequence and positional homologs foundin different strains, including sequences in any naturally occurring CMVstrain, and mutations to such strains as well as splice variants.

Gene sequences as well as ORFs and encoded polypeptides of differentstrains of CMV are known in the art including, without limitation, HCMVAD169 (American Type Culture Collection (ATCC) #VR 538), HCMV Towne(ATCC #VR 977), HCMV Davis (ATCC #VR 807), HCMV Toledo (Quinnan et al,Ann. Intern. Med., 101: 478-83 (1984)), monkey CMV Rh68.1 (ATCC #VR677), monkey CMV CSG (ATCC #VR 706), rat CMV Priscott (ATCC #VR 991),and mouse CMV Smith (ATCC #VR 1399). By way of a another example, geneand polypeptide sequence information of HCMV AD169 strain also isdescribed by GENBANK Accession Nos. BK000394.2 and X17403.1, each ofwhich is incorporated herein by reference in its entirety. Also, knownsequence information corresponding to one CMV strain (e.g., HCMV AD169strain) can be used to determine sequence information of genes andpolypeptides of another CMV strain. For example, homologs can bedetermined that includes genes sharing a common evolutional origin,structure/function, and the products of which encode polypeptides havingamino acid sequence identity of at least about 20%, illustratively,about 20 to about 100%, about 30 to about 90%, about 40 to about 80%,about 50 to about 70% sequence identity. A homolog can be identified bymethods known in the art such as comparison of the nucleic acid or aminoacid sequences to each other using computer programs, such as BLAST, orby hybridization under stringencies which are designed to detect apredetermined amount of mismatch between the sequences. Also, sequenceinformation based on homology can be employed to isolate andcharacterize sequences of a particular isolate, for example usingprimers and polymerase chain reaction (PCR).

In some embodiments, the at least one CMV antigen is a polypeptide, oran immunogenic fragment thereof, encoded by an open reading frame (ORF)of a HCMV gene or a homolog thereof. In one embodiment, the polypeptide,or the immunogenic fragment thereof, is encoded by a gene correspondingto the CMV strain shown by GENBANK Accession No. BK000394.2 or X17403.1.

In other embodiments, the at least one CMV antigen corresponds to apolypeptide, or an immunogenic fragment thereof, selected from the groupconsisting of: phosphoprotein unique long 83 (ppUL83; a/k/a pp65),glycoprotein UL55 (gpUL55; a/k/a gB), UL123 immediate early 1 (IE1)protein, UL122 IE2 protein, UL111A (a/k/a mtrII), US28, ppUL32, ppUL65,ppUL80a, ppUL82, ppUL98a, ppUL99, gpUL4 (a/k/a gp48), gpUL16, gpUL18(a/k/a MHC), gpUL75 (a/k/a gH), gpUL100, gpUL110 (a/k/a gM), gpUL115(a/k/a gL), pUL46, pUL48, pUL56, pUL86 (a/k/a MCP), glycoprotein uniqueshort 10 (gpUS10), gpUS11, glycoprotein complex II (gcII), gp65, andgp93.

In one embodiment, the at least one CMV antigen comprises an amino acidsequence corresponding to one or more epitopes from the same antigen ordistinct antigens of CMV. In some embodiments, the one or more epitopescan be characterized as restricted to or not restricted to a single MHCClass I haplotype. In other embodiments, the one or more epitopes isspecific for a sufficient number of MHC Class 1 molecules to providecoverage for at least about 5% of the general population,illustratively, for at least about 5, 10, 20, 30, 40, 50, 60, 70, 80,90, and 100% of the general population, irrespective of racial origin orethnicity. Those skilled in the art will readily be in a position todetermine the number of individual HCMV cytotoxic T lymphocytes (CTL)epitopes required to provide coverage of any given population using HLAspecificity experimentation. Optionally, the one or more epitopesfurther display HLA supertype specificity and/or comprise one or moreCD4+ determinants sufficient to facilitate a T-helper function in asubject.

Examples of CMV epitopes include, without limitation, the peptidescomprising the amino acid sequences as described by Trivedi et al.,Blood, 105:2793 (2005) and U.S. Patent Application Publication No.2005/0019344, each of which is incorporated herein by reference fortheir teaching of CMV epitopes. In one embodiment, the at least one CMVantigen is a polypeptide, or an antigenic fragment thereof, comprisingan amino acid sequence of SEQ ID NO:1, 2, or 3.

In another embodiment, the at least one antigen comprises one or moreCMV CTL epitopes restricted through dominant HLA alleles. For example, avaccination strategy can involve the generation of CD8+ T-cellrepertoire with formulations of synthetic peptides that mimicimmunodominant epitopes known to be recognized by a CMV-induced CTLresponse, for example by mixing multiple peptides or, alternatively, byusing minimal CTL epitopes that can be fused to construct a recombinantor synthetic polyepitope polypeptide. Also, a vaccine design based onCTL epitopes from latent antigens can be directed againstCMV-transformed latently infected cells as well as also be used inCMV-seronegative transplant recipients because of their increasedsusceptibility to CMV virus-induced conditions.

In other embodiments, the at least one antigen corresponds to a pool ofpeptides. In one embodiment, the pool of peptides comprises at leastabout 8-mer amino acid sequences, illustratively, about 8-mers to about30-mers, about 9-mers to about 25-mers, about 10-mers to about 20-mers,about 11-mers to about 18-mers, about 12-mers to about 16-mers, andabout 13-mers to about 15-mers, wherein the sequences of the peptideshave at least about 6 amino acids overlap, illustratively, about 6 toabout 20, about 7 to about 19, about 8 to about 18, about 9 to about 17,about 10 to about 16, and about 11 to about 14 amino acids overlap,wherein the pool of peptides covers at least about 10% of an HCMVprotein, illustratively, about 10 to about 100%, about 20 to about 90%,about 30 to about 80%, about 40 to about 70%, and about 50 to about 60%of the HCMV protein.

In one embodiment, the HCMV protein is an IE-1 protein (e.g., IE-1protein of HCMV strain AD169; see, e.g., Swiss-Prot Acc. No. P13202,which is herein incorporated by reference in its entirety). In anotherembodiment, the HCMV protein is a pp65 protein (e.g., pp65 protein ofHCMV strain AD169; see, e.g., Swiss-Prot Acc. No. P06725, which isherein incorporated by reference in its entirety).

In other embodiments, the pool of peptides comprises 15-mer amino acidsequences of 11 amino acids overlap, covering the complete orsubstantially complete sequence of one or more HCMV proteins. In someembodiment, the one or more HCMV proteins is an IE-1 protein or a pp65protein. For example, in some embodiments, the pool of peptidescomprises PepTivator™ CMV pp65 (Miltenyi Biotec, Gladbach, Germany) orPepTivator™ CMV IE-1 (Miltenyi Biotec, Gladbach, Germany).

In some embodiments, the use of peptide epitopes can make it easier toprepare products at current Good Manufacturing Practice (cGMP) grade.Thus, in some embodiments, the present invention provides an immunogeniccomposition comprising the pool of peptides.

Accordingly, in some aspects, the present invention provides one or moreimmunologically active peptides, and functional variants thereof,capable of eliciting a cellular immune response to a cell that expressesa CMV antigen. For example, the cell can be the cell can becharacterized as a cancer cell, a precancerous cell, or a cell-typepredisposed to developing cancer associated with CMV. In someembodiments, the peptides are capable of directing human CTL torecognize and lyse the cells. Such immunologically active peptides, inassociation with an MHC Class I molecule, can be recognized by CTL ofindividuals having a latent or active HCMV infection.

Such a peptide(s) (e.g., the pool of peptides) may be administered inthe form of a peptide(s) or lipopeptide(s) vaccine, optionally with anadjuvant. In some embodiments, the peptide(s) may be administered in theform of a cellular vaccine via the administration of autologous orallogeneic antigen presenting cells or dendritic cells that have beentreated in vitro so as to present a peptide of the pool on theirsurface. In another embodiment, T cells can be removed from anindividual and treated in vitro with the peptide(s), wherein theresulting CTL are reinfused autologously or allogeneically to thesubject. In various other embodiments, the peptide(s) of the presentinvention also may be administered to the subject, or in vitro to Tcells, in the form of a polynucleotide vaccine, wherein one or moresuitable gene transfer vectors, such as a plasmid or an engineered viralvector that contains DNA encoding the peptide fragment(s) under thecontrol of appropriate expression regulatory sequences, is administeredto the subject or to T cells in vitro.

Pools of peptides are disclosed by, e.g., Kiecker et al., Hum Immunol.65:523-36 (2204), which is herein incorporated by reference for itsteaching of pools of peptides and using pools of peptides forstimulating T cells.

The at least one CMV antigen also can be determined by routineexperimentation using techniques known in the art. For example,antigenic determinants of a particular CMV strain recognized by theimmune system can be cloned and characterized using libraries andexpression vectors. By way of example, random fragments of DNA can begenerated from a cosmid library of HCMV DNA. Fragments of variouslengths, e.g., about 50 to 600 bp in length, can be selected and clonedinto open reading frame (ORF) expression vectors to create ORF-librariesthat represent either the entire viral genome or defined subregions.Clones be isolated and screened immunologically for the synthesis offusion proteins consisting of an antigenic peptide encoded by the CMVsequence coupled to a reporter, e.g., an E. coli beta-galactosidasemolecule. Anti-CMV sera raised in animals as well as human hyperimmuneglobulin can be used for colony screening. Distinct sets of antigenicfusion proteins can be recognized by different antisera. Clones givingstrong reactions with immune sera can be mapped on the CMV genome andthe sequences of the CMV inserts determined. Antibodies against fusionproteins can be raised in mice or rabbits to identify the correspondingCMV proteins.

In other embodiments, the at least one CMV antigen further comprises oneor more signal sequences, for example, but not limited to, a gp96endoplasmic reticulum targeting peptide signal sequence, a LAMP-1lysosomal targeting peptide signal sequence, and the like. Signalsequences can enhance MHC class I and class II antigen processing andpresentation as well as provide other targeting properties. In otherembodiments, the at least one CMV antigen can be conjugated to a carrierprotein (e.g., Keyhole Limpet Hemocyanin (KLH), or synthesized as fusionproteins through recombination of nucleic acid coding sequences.

As further illustrated below, the pharmaceutically acceptablecomposition can comprise the at least one CMV antigen or nucleic acidsencoding the at least one CMV antigen.

A. Nucleic Acids

Generally, the subject can be inoculated with the pharmaceuticallyacceptable composition comprising nucleic acids through any parenteralroute. For example, the subject can be inoculated by intravenous,intraperitoneal, intradermal, subcutaneous, inhalation, or intramuscularroutes, or by particle bombardment using a gene gun. Preferably, muscletissue can be a site for the delivery and expression of polynucleotides.A dose of polynucleotides can be administered into muscle by multipleand/or repetitive injections, for example, to extend administration overlong periods of time. Thus, muscle cells can be injected withpolynucleotides coding for the at least one CMV antigen, and theexpressed antigens can be presented by muscle cells in the context ofantigens of the major histocompatibility complex to elicit the immuneresponse against the at least one CMV antigen.

The epidermis can be another useful site for the delivery and expressionof polynucleotides, for example either by direct injection or particlebombardment. A dose of polynucleotides can be administered in theepidermis, for example by multiple injections or bombardments to extendadministering over long periods of time. For example, skin cells can beinjected with polynucleotides coding for the at least one CMV antigen,and the expressed antigens can be presented by skin cells in the contextof antigens of the major histocompatibility complex to elicit the immuneresponse against the at least one CMV antigen.

A subject also can be inoculated by a mucosal route. The polynucleotidescan be administered to a mucosal surface by a variety of methodsincluding polynucleotide-containing nose-drops, inhalants,suppositories, microsphere-encapsulated polynucleotides, or bybombardment with polynucleotide-coated gold particles. For example, thenucleic acids coding for the at least one CMV antigen can beadministered to a respiratory mucosal surface.

Any appropriate physiologically compatible medium, such as saline forinjection, or gold particles for particle bombardment, is suitable forintroducing polynucleotides into a subject.

1. RNA

In some embodiments, the pharmaceutically acceptable compositioncomprises nucleic acids encoding the at least one CMV antigen, whereinthe nucleic acids are RNA. The RNAs comprise translatable RNA templatesto guide the intracellular synthesis of amino acid chains that providethe at least one CMV antigen. RNAs encoding the at least one CMV antigenalso can be in vitro transcribed, e.g., reverse transcribed to producecDNAs that can then be amplified by PCR, if desired, and subsequentlytranscribed in vitro, with or without cloning the cDNA. A number ofmethods are available to one of ordinary skill in the art to prepareRNAs encoding the at least one CMV antigen. Thus, for example,conventional in vitro transcription techniques and bacterial polymerasescan be used to produce in vitro transcribed RNAs, or the in vitrotranscribed RNAs can be synthesized from cloned DNA sequences encodingthe at least one CMV antigen.

2. DNA

In another embodiment, the nucleic acids encoding the at least one CMVantigen comprise DNAs having open reading frames encoding the at leastone CMV antigen. For example, a pharmaceutically acceptable compositioncomprising expression vectors having DNA open reading frames encodingthe at least one CMV antigen can be administered to the subject.

Genomic DNA fragments and/or cDNAs comprising open reading framesencoding the at least one CMV antigen can be employed in the methods ofthe present invention. cDNAs can be prepared from the above-describedRNAs coding for the at least one CMV antigen using techniques known toone of ordinary skill in the art. If desired, DNA can be fragmented, forexample by physical fragmentation or, preferably, by enzymatic cleavage,i.e. use of restriction endonucleases. Fragmentation methods are wellknown to those skilled in the art and can be varied (e.g., by use ofdifferent restriction endonucleases or combinations and digestion times)to obtain fragments differing in size and composition. DNAs or fragmentsthereof having open reading frames encoding the at least one CMV antigencan be cloned into expression vectors by methods and reagents known inthe art.

Standard cloning vectors can be employed that have a selectable marker(e.g., ampicillin) and, preferably an origin of replication (e.g., ori)and a suitable promoter. Bacteria (e.g., E. coli) or other suitable hostcan then transformed with the vectors, and transformants cultured bystandard procedures and the plasmid DNA isolated by such methods aschromatographic or organic separation. For example, plasmids areavailable for cloning into a site which can direct the at least one CMVantigen expressed by the open reading frames to MHC I or II. Expressionvectors used for eliciting an immune response and methods of using sameare described in U.S. Patent Application Publication No. 20040241140,which is incorporated herein for its teaching of expression vectors usedfor eliciting an immune response and methods of using same.

When taken up by a cell (e.g., muscle cell, an antigen-presenting cell(APC) such as a dendritic cell, macrophage, etc.), a DNA molecule can bepresent in the cell as an extrachromosomal molecule and/or can integrateinto the chromosome. DNA can be introduced into cells in the form of aplasmid which can remain as separate genetic material. Alternatively,linear DNAs that can integrate into the chromosome can be introducedinto the cell. Optionally, when introducing DNA into a cell, reagentswhich promote DNA integration into chromosomes can be added.

Thus, preferably DNAs include regulatory elements necessary forexpression of an open reading frame. Such elements can include, forexample, a promoter, an initiation codon, a stop codon, and apolyadenylation signal. In addition, enhancers can be included. As isknown in the art, these elements are preferably operably linked to asequence that encodes the at least one CMV antigen. Regulatory elementsare preferably selected that are operable in the species of the subjectto which they are to be administered. Initiation codons and stop codonsin frame with a coding sequence are preferably included.

Examples of promoters include but are not limited to promoters fromSimian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, HumanImmunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR)promoter, Moloney virus, Cytomegalovirus (CMV) such as the CMV immediateearly promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) aswell as promoters from human genes such as human actin, human myosin,human hemoglobin, human muscle creatine, and human metalothionein.Examples of suitable polyadenylation signals include but are not limitedto SV40 polyadenylation signals and LTR polyadenylation signals.

In addition to the regulatory elements required for DNA expression,other elements may also be included in the DNA molecule. Such additionalelements include enhancers. Enhancers include the promoters describedhereinabove. Preferred enhancers/promoters include, for example, humanactin, human myosin, human hemoglobin, human muscle creatine and viralenhancers such as those from CMV, RSV and EBV.

Optionally, the DNAs can be operably incorporated in a carrier ordelivery vector. Useful delivery vectors include but are not limited tobiodegradable microcapsules, immuno-stimulating complexes (ISCOMs) orliposomes, and genetically engineered attenuated live carriers such asviruses or bacteria.

In some embodiments, the vector is a viral vector, such as lentiviruses,retroviruses, herpes viruses, adenoviruses, adeno-associated viruses,vaccinia viruses, baculoviruses, Fowl pox, AV-pox, modified vacciniaAnkara (MVA) and other recombinant viruses. For example, a vacciniavirus vector can be used to infect dendritic cells.

Thus, a vector encoding the at least one CMV antigen or an immunogenicfragment thereof can be introduced in vivo, ex vivo, or in vitro using aviral vector or through direct introduction of DNA. Expression intargeted tissues can be effected by targeting the transgenic vector tospecific cells, such as with a viral vector or a receptor ligand, or byusing a tissue-specific promoter, or both.

Viral vectors commonly used for in vivo or ex vivo targeting andvaccination procedures are DNA-based vectors and retroviral vectors.Methods for constructing and using viral vectors are known in the art(see, e.g., Miller and Rosman, BioTechniques, 7:980-990, 1992).Preferably, the viral vectors are replication defective, i.e., they areunable to replicate autonomously in the target cell. Preferably, thereplication defective virus is a minimal virus, i.e., it retains onlythe sequences of its genome which are necessary for encapsidating thegenome to produce viral particles.

DNA viral vectors include an attenuated or defective DNA virus, such asbut not limited to herpes simplex virus (HSV), papillomavirus, EpsteinBarr virus (EBV), adenovirus, adeno-associated virus (AAV), vacciniavirus, and the like. Examples of particular vectors include, but are notlimited to, a defective herpes virus 1 (HSV1) vector (Kaplitt, et al.,Molec. Cell. Neurosci. 2:320-330, 1991; International Patent PublicationNo. WO 94/21807, published Sep. 29, 1994; International PatentPublication No. WO 92/05263, published Apr. 2, 1994); an attenuatedadenovirus vector, such as the vector described byStratford-Perricaudet, et al. (J. Clin. Invest. 90:626-630, 1992; seealso La Salle, et al., Science 259:988-990, 1993); and a defectiveadeno-associated virus vector (Samulski, et al., J. Virol. 61:3096-3101,1987; Samulski, et al., J. Virol. 63:3822-3828, 1989; Lebkowski, et al.,Mol. Cell. Biol. 8:3988-3996, 1988). Viral vectors also are commerciallyavailable.

Optionally, the DNAs also can be provided with reagents that improve theuptake of the genetic material by cells. For example, the DNA can beformulated with or administered in conjunction with an uptakefacilitator reagent selected from the group consisting of benzoic acidesters, anilides, amidines, and urethans.

B. Dendritic Cells

One of ordinary skill in the art will recognize that the capacity togenerate dendritic cells (DCs) in vitro also can be used in accordancewith the present invention for ex vivo loading of DCs with the at leastone CMV antigen, or nucleic acids encoding the at least one CMV antigen,and administration of DC vaccines as a strategy for eliciting an immuneresponse to a cell that expresses a CMV antigen. Preclinical studieshave shown DCs to be potent activators of de novo and recall responsesin B and T lymphocytes.

In some embodiments, the present invention provides a method foreliciting in a subject an immune response to a cell that expresses aCMV, the method comprising administering to the subject apharmaceutically acceptable composition comprising dendritic cellsloaded ex vivo with at least one CMV antigen, or nucleic acids encodingthe at least one CMV antigen, wherein the pharmaceutically acceptablecomposition, when administered to the subject, elicits the immuneresponse to the cell.

Accordingly, the CMV antigen-primed antigen-presenting cells of thepresent invention and the CMV antigen-specific T lymphocytes generatedwith these antigen-presenting cells can be used as active compounds inimmunomodulating compositions for prophylactic or therapeuticapplications. As described below, in some embodiments, theantigen-primed antigen-presenting cells of the invention can be used forgenerating cytotoxic T lymphocytes (CTL) (e.g., CD8+ or CD4+ CTL) foradoptive transfer to the subject.

C. Compositions

In other aspects, the present invention provides a pharmaceuticallyacceptable composition comprising at least one CMV antigen or nucleicacids encoding the at least one CMV antigen. The pharmaceuticallyacceptable composition, when administered to a subject, can elicit animmune response against a cell that expresses a CMV antigen. Thepharmaceutically acceptable compositions of the present invention can beuseful as vaccine compositions for prophylactic or therapeutic treatmentof a neoplastic disease or symptoms thereof, particularly for preventingor treating CMV-associated cancer (e.g., a tumor) in the subject.

In some embodiments, the pharmaceutically acceptable composition furthercomprises a physiologically acceptable carrier, diluent, or excipient.Techniques for formulating and administering also can be found inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,latest edition.

Pharmaceutically acceptable carriers known in the art include, but arenot limited to, sterile water, saline, glucose, dextrose, or bufferedsolutions. Agents such as diluents, stabilizers (e.g., sugars and aminoacids), preservatives, wetting agents, emulsifying agents, pH bufferingagents, additives that enhance viscosity, and the like. Preferably, themedium or carrier will produce minimal or no adverse effects.

In other embodiments, the pharmaceutically acceptable compositionfurther comprises a physiologically acceptable adjuvant. Preferably, theadjuvant employed provides for increased immunogenicity. The adjuvantcan be one that provides for slow release of antigen (e.g., the adjuvantcan be a liposome), or it can be an adjuvant that is immunogenic in itsown right thereby functioning synergistically with antigens. Forexample, the adjuvant can be a known adjuvant or other substance thatpromotes nucleic acid uptake, recruits immune system cells to the siteof administration, or facilitates the immune activation of respondinglymphoid cells. Adjuvants include, but are not limited to,immunomodulatory molecules (e.g., cytokines), oil and water emulsions,aluminum hydroxide, glucan, dextran sulfate, iron oxide, sodiumalginate, Bacto-Adjuvant, synthetic polymers such as poly amino acidsand co-polymers of amino acids, saponin, paraffin oil, and muramyldipeptide.

In one embodiment, the adjuvant is an immunomodulatory molecule. Forexample, the immunomodulatory molecule can be a recombinant proteincytokine, chemokine, or immunostimulatory agent or nucleic acid encodingcytokines, chemokines, or immunostimulatory agents designed to enhancethe immunologic response.

Examples of immunomodulatory cytokines include interferons (e.g., IFNα,IFNβ and IFNγ), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-12 and IL-20), tumor necrosis factors (e.g.,TNFα and TNFβ), erythropoietin (EPO), FLT-3 ligand, gIp10, TCA-3, MCP-1,MIF, MIP-1α, MIP-1β, Rantes, macrophage colony stimulating factor(M-CSF), granulocyte colony stimulating factor (G-CSF), andgranulocyte-macrophage colony stimulating factor (GM-CSF), as well asfunctional fragments of any of the foregoing. Any immunomodulatorychemokine that binds to a chemokine receptor, i.e., a CXC, CC, C, orCX3C chemokine receptor, also can be used in the context of the presentinvention. Examples of chemokines include, but are not limited to,Mip1α, Mip-1β, Mip-3α (Larc), Mip-3β, Rantes, Hcc-1, Mpif-1, Mpif-2,Mcp-1, Mcp-2, Mcp-3, Mcp-4, Mcp-5, Eotaxin, Tarc, Elc, I309, IL-8, Gcp-2Gro-α, Gro-β, Gro-γ, Nap-2, Ena-78, Gcp-2, Ip-10, Mig, I-Tac, Sdf-1, andBca-1 (Blc), as well as functional fragments of any of the foregoing.

In another embodiment, the adjuvant is a cytokine selected from thegroup consisting of: GM-CSF, G-CSF, IL-2, IL-4, IL-7, IL-12, IL-15,IL-21, TNF-α, and M-CSF. In some embodiments, the adjuvant is comprisedof incomplete Freund's adjuvant (Montanide ISA 51) or Corynebacteriumgranulosum P40.

One of ordinary skill in the art will appreciate that some of theseadjuvants cannot be expressed from a vector, in which case the adjuvant,when used, can be administered simultaneously or sequentially, in anyorder.

One of ordinary skill in the art knows that methods and compositions ofthe present invention can be used as part of combination therapies, forexample as methods and/or compositions comprising one or more otheragents such as, but not limited to, chemotherapeutic, immunotherapeutic,immunomodulatory, anti-angiogenic, anti-viral agents, and hormonalagents.

Examples of anti-viral agents include, but are not limited, aganciclovir (e.g., CYTOVENE®), a valganciclovir (e.g., Valcyte®), afoscarnet (e.g., FOSCAVIR®), a cidofovir (e.g., VISTIDE®, HPMPC), anadefovir (e.g., PMEA, PREVEON®, HEPSERA®), an acyclovir (e.g.,ZOVIRAX®), a valacyclovir (e.g., VALTREX™, ZELITREX™), a polyanion, anda protein kinase C inhibitor (e.g., a bis-indolylmaleide). In oneembodiment, the anti-viral agent employed in combination with thecompositions and methods of the present invention is a ganciclover, avalganciclovir, a cidofovir, or a foscarnet.

In other embodiments, the one or more other agents effect CMV antigenexpression, preferably by enhancing CMV antigen expression, preferablyby enhancing CMV antigen expression in latently infected cells,preferably in glioma cells. In one embodiment, the one or more agents isselected from the group consisting of: a retinoic acid (RA), alemtuzumb(Campath 1H), and an immunosuppressive agent (e.g., cyclophosphamide,cyclosporine). In another embodiment, irradiation and/or immunodeleptionis provided in combination with the compositions and methods of thepresent invention.

In various embodiments, the one or more other agents can be achemotherapeutic agent, naturally occurring or synthetic, for example asdescribed in “Cancer Chemotherapeutic Agents”, American ChemicalSociety, 1995, W. O. Foye Ed.

In one embodiment, the chemotherapeutic agent is selected from the groupconsisting of a small molecule receptor antagonists such as vatalanib,SU 11248 or AZD-6474, EGFR or HER2 antagonists such as gefitinib,erlotinib, CI-1033 or Herceptin, antibodies such as bevacizumab,cetuximab, rituximab, DNA alkylating drugs such as cisplatin,oxaliplatin or carboplatin, anthracyclines such as doxorubicin orepirubicin, an antimetabolite such as 5-FU, pemetrexed, gemcitabine orcapecitabine, a camptothecin such as irinotecan or topotecan, ananti-cancer drug such as paclitaxel or docetaxel, an epipodophyllotoxinsuch as etoposide or teniposide, a proteasome inhibitor such asbortezomib or anti-inflammatory drugs such as celecoxib or rofecoxib,optionally in form of the pharmaceutically acceptable salts, in form ofthe hydrates and/or solvates and optionally in the form of theindividual optical isomers, mixtures of the individual enantiomers orracemates thereof.

In another embodiment, the chemotherapeutic agent is selected from thegroup consisting of a small molecule VEGF receptor antagonist such asvatalanib (PTK-787/ZK222584), SU-5416, SU-6668, SU-11248, SU-14813,AZD-6474, AZD-2171, CP-547632, CEP-7055, AG-013736, IM-842 or GW-786034,a dual EGFR/HER2 antagonist such as gefitinib, erlotinib, CI-1033 orGW-2016, an EGFR antagonist such as iressa (ZD-1839), tarceva (OSI-774),PKI-166, EKB-569, HKI-272 or herceptin, an antagonist of themitogen-activated protein kinase such as BAY-43-9006 or BAY-57-9006, aquinazoline derivative such as4-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(N,N-dimethylamino)-1-oxo-2-bute-n-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)quinazolineor4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-(homomorpholin-4-yl)-1-oxo-2-bu-ten-1-yl]amino}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline,or a pharmaceutically acceptable salt thereof, a protein kinase receptorantagonist which is not classified under the synthetic small moleculessuch as atrasentan, rituximab, cetuximab, Avastin™ (bevacizumab),IMC-1C11, erbitux (C-225), DC-101, EMD-72000, vitaxin, imatinib, aprotein tyrosine kinase inhibitor which is a fusion protein such asVEGFtrap, an alkylating agent or a platinum compound such as melphalan,cyclophosphamide, an oxazaphosphorine, cisplatin, carboplatin,oxaliplatin, satraplatin, tetraplatin, iproplatin, mitomycin,streptozocin, carmustine (BCNU), lomustine (CCNU), busulfan, ifosfamide,streptozocin, thiotepa, chlorambucil, a nitrogen mustard such asmechlorethamine, an ethyleneimine compound, an alkylsulphonate,daunorubicin, doxorubicin (adriamycin), liposomal doxorubicin (doxil),epirubicin, idarubicin, mitoxantrone, amsacrine, dactinomycin,distamycin or a derivative thereof, netropsin, pibenzimol, mitomycin,CC-1065, a duocarmycin, mithramycin, chromomycin, olivomycin, aphtalanilide such as propamidine or stilbamidine, an anthramycin, anaziridine, a nitrosourea or a derivative thereof, a pyrimidine or purineanalogue or antagonist or an inhibitor of the nucleoside diphosphatereductase such as cytarabine, 5-fluorouracile (5-FU), pemetrexed,tegafur/uracil, uracil mustard, fludarabine, gemcitabine, capecitabine,mercaptopurine, cladribine, thioguanine, methotrexate, pentostatin,hydroxyurea, or folic acid, a phleomycin, a bleomycin or a derivative orsalt thereof, CHPP, BZPP, MTPP, BAPP, liblomycin, an acridine or aderivative thereof, a rifamycin, an actinomycin, adramycin, acamptothecin such as irinotecan (camptosar) or topotecan, an amsacrineor analogue thereof, a tricyclic carboxamide, an histonedeacetylaseinhibitor such as SAHA, MD-275, trichostatin A, CBHA, LAQ824, orvalproic acid, an anti-cancer drug from plants such as paclitaxel(taxol), docetaxel or taxotere, a vinca alkaloid such as navelbine,vinblastin, vincristin, vindesine or vinorelbine, a tropolone alkaloidsuch as colchicine or a derivative thereof, a macrolide such asmaytansine, an ansamitocin or rhizoxin, an antimitotic peptide such asphomopsin or dolastatin, an epipodophyllotoxin or a derivative ofpodophyllotoxin such as etoposide or teniposide, a steganacin, anantimitotic carbamate derivative such as combretastatin or amphetinile,procarbazine, a proteasome inhibitor such as bortezomib, an enzyme suchas asparaginase, pegylated asparaginase (pegaspargase) or athymidine-phosphorylase inhibitor, a gestagen or an estrogen such asestramustine (T-66) or megestrol, an anti-androgen such as flutamide,casodex, anandron or cyproterone acetate, an aromatase inhibitor such asaminogluthetimide, anastrozole, formestan or letrozole, a GNrH analoguesuch as leuprorelin, buserelin, goserelin or triptorelin, ananti-estrogen such as tamoxifen or its citrate salt, droloxifene,trioxifene, raloxifene or zindoxifene, a derivative of 17β-estradiolsuch as ICI 164,384 or ICI 182,780, aminoglutethimide, formestane,fadrozole, finasteride, ketoconazole, a LH-RH antagonist such asleuprolide, a steroid such as prednisone, prednisolone,methylprednisolone, dexamethasone, budenoside, fluocortolone ortriamcinolone, an interferon such as interferon β, an interleukin suchas IL-10 or IL-12, an anti-TNFα antibody such as etanercept, animmunomodulatory drug such as thalidomide, its R- and S-enantiomers andits derivatives, or revimid (CC-5013), a leukotrien antagonist,mitomycin C, an aziridoquinone such as BMY-42355, AZQ or EO-9, a2-nitroimidazole such as misonidazole, NLP-1 or NLA-1, a nitroacridine,a nitroquinoline, a nitropyrazoloacridine, a “dual-function” nitroaromatic such as RSU-1069 or RB-6145, CB-1954, a N-oxide of nitrogenmustard such as nitromin, a metal complex of a nitrogen mustard, ananti-CD3 or anti-CD25 antibody, a tolerance induction agent, abiphosphonate or derivative thereof such as minodronic acid or itsderivatives (YM-529, Ono-5920, YH-529), zoledronic acid monohydrate,ibandronate sodium hydrate or clodronate disodium, a nitroimidazole suchas metronidazole, misonidazole, benznidazole or nimorazole, a nitroarylcompound such as RSU-1069, a nitroxyl or N-oxide such as SR-4233, anhalogenated pyrimidine analogue such as bromodeoxyuridine,iododeoxyuridine, a thiophosphate such as WR-272 1, a photo-chemicallyactivated drug such as porfimer, photofrin, a benzoporphyrin derivative,a pheophorbide derivative, merocyanin 540 (MC-540) or tin etioporpurin,an ant-template or an anti-sense RNA or DNA such as oblimersen, anon-steroidal inflammatory drug such as acetylsalicyclic acid,mesalazin, ibuprofen, naproxen, flurbiprofen, fenoprofen, fenbufen,ketoprofen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen,miroprofen, tioxaprofen, suprofen, alminoprofen, tiaprofenic acid,fluprofen, indomethacin, sulindac, tolmetin, zomepirac, nabumetone,diclofenac, fenclofenac, alclofenac, bromfenac, ibufenac, aceclofenac,acemetacin, fentiazac, clidanac, etodolac, oxpinac, mefenamic acid,meclofenamic acid, flufenamic acid, nifluminic acid, tolfenamic acid,diflunisal, flufenisal, piroxicam, tenoxicam, lomoxicam, nimesulide,meloxicam, celecoxib, rofecoxib, or a pharmaceutically acceptable saltof a non-steroidal inflammatory drug, a cytotoxic antibiotic, anantibody targeting the surface molecules of cancer cells such asapolizumab or 1D09C3, an inhibitor of metalloproteinases such as TIMP-1or TIMP-2, Zinc, an inhibitor of oncogenes such as P53 and Rb, a complexof rare earth elements such as the heterocyclic complexes oflanthanides, a photo-chemotherapeutic agent such as PUVA, an inhibitorof the transcription factor complex ESX/DRIP130/Sur-2, an inhibitor ofHER-2 expression, such as the heat shock protein HSP90 modulatorgeldanamycin and its derivative 17-allylaminogeldanamycin or 17-AAG, ora therapeutic agent selected from IM-842, tetrathiomolybdate,squalamine, combrestatin A4, TNP-470, marimastat, neovastat,bicalutamide, abarelix, oregovomab, mitumomab, TLK-286, alemtuzumab,ibritumomab, temozolomide, denileukin diftitox, aldesleukin,dacarbazine, floxuridine, plicamycin, mitotane, pipobroman, plicamycin,tamoxifen and testolactone. Preferred compounds include small moleculeVEGF receptor antagonist such as vatalanib (PTK-787/ZK222584), SU-5416,SU-6668, SU-11248, SU-14813, AZD-6474, EGFR/HER2 antagonists such asCI-1033 or GW-2016, an EGFR antagonist such as iressa (gefitinib,ZD-1839), tarceva (erlotinib, OSI-774), PKI-166, EKB-569, HKI-272 orherceptin, an antagonist of the mitogen-activated protein kinase such asBAY-43-9006 or BAY-57-9006, atrasentan, rituximab, cetuximab, Avastin™(bevacizumab), IMC-1C11, erbitux (C-225), DC-101, EMD-72000, vitaxin,imatinib, an alkylating agent or a platinum compound such as melphalan,cyclophosphamide, cisplatin, carboplatin, oxaliplatin, satraplatin,daunorubicin, doxorubicin (adriamycin), liposomal doxorubicin (doxil),epirubicin, idarubicin, a pyrimidine or purine analogue or antagonist oran inhibitor of the nucleoside diphosphate reductase such as cytarabine,5-fluorouracile (5-FU), pemetrexed, tegafur/uracil, gemcitabine,capecitabine, mercaptopurine, methotrexate, an anti-cancer drug such aspaclitaxel (taxol) or docetaxel, a vinca alkaloid such as navelbine,vinblastin, vincristin, vindesine or vinorelbine, an antimitotic peptidesuch as dolastatin, an epipodophyllotoxin or a derivative ofpodophyllotoxin such as etoposide or teniposide, a non-steroidalinflammatory drug such as meloxicam, celecoxib, rofecoxib, an antibodytargeting the surface molecules of cancer cells such as apolizumab orID09C3 or the heat shock protein HSP90 modulator geldanamycin and itsderivative 17-allylaminogeldanamycin or 17-AAG.

In another embodiment, the chemotherapeutic agent is selected from thegroup consisting of compounds interacting with or binding tubulin,synthetic small molecule VEGF receptor antagonists, small moleculegrowth factor receptor antagonists, inhibitors of the EGF receptorand/or VEGF receptor and/or integrin receptors or any other proteintyrosine kinase receptors which are not classified under the syntheticsmall-molecules, inhibitors directed to EGF receptor and/or VEGFreceptor and/or integrin receptors or any other protein tyrosine kinasereceptors, which are fusion proteins, compounds which interact withnucleic acids and which are classified as alkylating agents or platinumcompounds, compounds which interact with nucleic acids and which areclassified as anthracyclines, as DNA intercalators or as DNAcross-linking agents, including DNA minor-groove binding compounds,anti-metabolites, naturally occurring, semi-synthetic or syntheticbleomycin type antibiotics, inhibitors of DNA transcribing enzymes, andespecially the topoisomerase I or topoisomerase II inhibitors, chromatinmodifying agents, mitosis inhibitors, anti-mitotic agents, cell-cycleinhibitors, proteasome inhibitors, enzymes, hormones, hormoneantagonists, hormone inhibitors, inhibitors of steroid biosynthesis,steroids, cytokines, hypoxia-selective cytotoxins, inhibitors ofcytokines, lymphokines, antibodies directed against cytokines, oral andparenteral tolerance induction agents, supportive agents, chemicalradiation sensitizers and protectors, photo-chemically activated drugs,synthetic poly- or oligonucleotides, optionally modified or conjugated,non-steroidal anti-inflammatory drugs, cytotoxic antibiotics, antibodiestargeting the surface molecules of cancer cells, antibodies targetinggrowth factors or their receptors, inhibitors of metalloproteinases,metals, inhibitors of oncogenes, inhibitors of gene transcription or ofRNA translation or protein expression, complexes of rare earth elements,and photo-chemotherapeutic agents.

In other embodiments, the chemotherapeutic agent is selected from thegroup consisting of paclitaxel (taxol), docetaxel, a vinca alkaloid suchas navelbine, vinblastin, vincristin, vindesine or vinorelbine, analkylating agent or a platinum compound such as melphalan,cyclophosphamide, an oxazaphosphorine, cisplatin, carboplatin,oxaliplatin, satraplatin, tetraplatin, iproplatin, mitomycin,streptozocin, carmustine (BCNU), lomustine (CCNU), busulfan, ifosfamide,streptozocin, thiotepa, chlorambucil, a nitrogen mustard such asmechlorethamine, an immunomodulatory drug such as thalidomide, its R-and S-enantiomers and its derivatives, or revimid (CC-5013)), anethyleneimine compound, an alkylsulphonate, daunorubicin, doxorubicin(adriamycin), liposomal doxfflubicin (doxil), epirubicin, idarubicin,mitoxantrone, amsacrine, dactinomycin, distamycin or a derivativethereof, netropsin, pibenzimol, mitomycin, CC-1065, a duocarmycin,mithramycin, chromomycin, olivomycin, a phtalanilide such as propamidineor stilbamidine, an anthramycin, an aziridine, a nitrosourea or aderivative thereof, a pyrimidine or purine analogue or antagonist or aninhibitor of the nucleoside diphosphate reductase such as cytarabine,5-fluorouracile (5-FU), uracil mustard, fludarabine, gemcitabine,capecitabine, mercaptopurine, cladribine, thioguanine, methotrexate,pentostatin, hydroxyurea, or folic acid, an acridine or a derivativethereof, a rifamycin, an actinomycin, adramycin, a camptothecin such asirinotecan (camptosar) or topotecan, an amsacrine or analogue thereof, atricyclic carboxamide, an histonedeacetylase inhibitor such as SAHA,MD-275, trichostatin A, CBHA, LAQ824, or valproic acid, a proteasomeinhibitor such as bortezomib, a small molecule VEGF receptor antagonistsuch as vatalanib (PTK-787/ZK222584), SU-5416, SU-6668, SU-11248,SU-14813, AZD-6474, AZD-2171, CP-547632, CEP-7055, AG-013736, IM-842 orGW-786034, an antagonist of the mitogen-activated protein kinase such asBAY-43-9006 or BAY-57-9006, a dual EGFR/HER2 antagonist such asgefitinib, erlotinib, CI-1033 or GW-2016, an EGFR antagonist such asiressa (ZD-1839), tarceva (OSI-774), PKI-166, EKB-569, HKI-272 orherceptin, a quinazoline derivative such as4-[(3-chloro-4-fluorophenyl)amino]-6-{[-4-(N,N-dimethylamino)-1-oxo-2-but-en-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)-quinazolineor4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-(homomorpholin-4-yl)-1-oxo-2-bu-ten-1-yl]amino-}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline,or a pharmaceutically acceptable salt thereof, an inhibitor of thetranscription factor complex ESX/DRIP130/Sur-2, an inhibitor of HER-2expression, such as the heat shock protein HSP90 modulator geldanamycinand its derivative 17-allylaminogeldanamycin or 17-AAG, a protein kinasereceptor antagonist which is not classified under the synthetic smallmolecules such as atrasentan, rituximab, cetuximab, Avastin™(bevacizumab), IMC-1C11, erbitux (C-225), DC-101, EMD-72000, vitaxin,imatinib, and an antibody targeting the surface molecules of cancercells such as apolizumab or 1D09C3.

In some other embodiments, the chemotherapeutic agent is a compoundwhich reduces the transport of hyaluronan mediated by one or more ABCtransporters, or drug transport inhibitor, such as a P-glycoprotein(P-gp) inhibitor molecule or inhibitor peptide, an MRP1 inhibitor, anantibody directed against and capable of blocking the ABC transporter,an antisense oligomer, iRNA, siRNA or aptamer directed against one ormore ABC transporters. Examples of P-glycoprotein (P-gp) inhibitormolecules in accordance with the present invention are zosuquidar (LY335973), its salts (especially the trichloride salt) and its polymorphs,cyclosporin A (also known as cyclosporine), verapamil or its R-isomer,tamoxifen, quinidine, d-alpha tocopheryl polyethylene glycol 1000succinate, VX-710, PSC833, phenothiazine, GF120918 (II), SDZ PSC 833,TMBY, MS-073, S-9788, SDZ 280-446, XR(9051) and functional derivatives,analogues and isomers of these.

D. Adoptive Immunotherapy

In other aspects, the at least one CMV antigen, or nucleic acidsencoding the at least one CMV antigen, also can provide for compositionsand methods for providing CMV antigen-primed, antigen-presenting cells,and/or CMV antigen-specific T lymphocytes generated with theseantigen-presenting cells, e.g., for use as active compounds inimmunomodulating compositions and methods for prophylactic ortherapeutic applications directed at cells that express a CMV antigen.

Accordingly, in one aspect, the invention provides a method for makingCMV antigen-primed, antigen-presenting cells by:

contacting antigen-presenting cells with at least one CMV antigen, ornucleic acids encoding the at least one CMV antigen, in vitro under acondition sufficient for the at least one CMV antigen to be presented bythe antigen-presenting cells. The at least one CMV antigen, or nucleicacids encoding the at least one CMV antigen, are as described above.

The at least one CMV antigen, or nucleic acids encoding the at least oneCMV antigen, can be contacted with a homogenous, substantiallyhomogenous, or heterogeneous composition comprising antigen-presentingcells. For example, the composition can include but is not limited towhole blood, fresh blood, or fractions thereof such as, but not limitedto, peripheral blood mononuclear cells, buffy coat fractions of wholeblood, packed red cells, irradiated blood, dendritic cells, monocytes,macrophages, neutrophils, lymphocytes, natural killer cells, and naturalkiller T cells. If, optionally, precursors of antigen-presenting cellsare used, the precursors can be cultured under suitable cultureconditions sufficient to differentiate the precursors intoantigen-presenting cells. Preferably, the antigen-presenting cells (or,optionally, precursors) are selected from monocytes, macrophages, cellsof myeloid lineage, B cells, dendritic cells, or Langerhans cells.

The amount of the at least one CMV antigen, or nucleic acids encodingthe at least one CMV antigen, to be placed in contact withantigen-presenting cells can be determined by one of ordinary skill inthe art by routine experimentation. Generally, antigen-presenting cellsare contacted with the at least one CMV antigen, or nucleic acidsencoding the at least one CMV antigen, for a period of time sufficientfor cells to present the processed forms of the antigens for themodulation of T cells. In one embodiment, antigen-presenting cells areincubated in the presence of the at least one CMV antigen, or nucleicacids encoding the at least one CMV antigen, for less than about a week,illustratively, for about 1 minute to about 48 hours, about 2 minutes toabout 36 hours, about 3 minutes to about 24 hours, about 4 minutes toabout 12 hours, about 6 minutes to about 8 hours, about 8 minutes toabout 6 hours, about 10 minutes to about 5 hours, about 15 minutes toabout 4 hours, about 20 minutes to about 3 hours, about 30 minutes toabout 2 hours, and about 40 minutes to about 1 hour. The time and amountof antigens, or nucleic acids encoding the antigens, necessary for theantigen presenting cells to process and present the antigens can bedetermined, for example using pulse-chase methods wherein contact isfollowed by a washout period and exposure to a read-out system e.g.,antigen reactive T cells.

Typically, the length of time necessary for an antigen-presenting cellto present an antigen on its surface can vary depending on a number offactors including the antigen or form (e.g., peptide versus encodingpolynucleotide) of antigen employed, its dose, and theantigen-presenting cell employed, as well as the conditions under whichantigen loading is undertaken. These parameters can be determined by theskilled artisan using routine procedures. Efficiency of priming of anantigen-presenting cell can be determined by assaying T cell cytotoxicactivity in vitro or using antigen-presenting cells as targets of CTLs.Other methods that can detect the presence of antigen on the surface ofantigen-presenting cells are also contemplated by the presentedinvention.

A number of methods for delivery of antigens to the endogenousprocessing pathway of antigen-presenting cells are known. Such methodsinclude but are not limited to methods involving pH-sensitive liposomes,coupling of antigens to potent adjuvants, apoptotic cell delivery,pulsing cells onto dendritic cells, delivering recombinant chimericvirus-like particles (VLPs) comprising antigen to the MHC class Iprocessing pathway of a dendritic cell line.

In one embodiment, solubilized CMV antigen is incubated withantigen-presenting cells. In other embodiments, the at least one CMVantigen can be coupled to a cytolysin to enhance the transfer of theantigens into the cytosol of an antigen-presenting cell for delivery tothe MHC class I pathway. Exemplary cytolysins include saponin compoundssuch as saponin-containing Immune Stimulating Complexes (ISCOMs),pore-forming toxins (e.g., an alpha-toxin), and natural cytolysins ofgram-positive bacteria such as listeriolysin O (LLO), streptolysin O(SLO), and perfringolysin O (PFO).

By way of another example, in other embodiments, antigen-presentingcells, preferably dendritic cells and macrophage, can be isolatedaccording to methods known in the art and transfected withpolynucleotides by methods known in the art for introducing nucleicacids encoding CMV antigens into the APCs. Transfection reagents andmethods (e.g., SuperFect®) also are commercially available. For example,RNAs encoding CMV antigens can be provided in a suitable medium (e.g.,Opti-MEM®) and combined with a lipid (e.g., a cationic lipid) prior tocontact with APCs. Non-limiting examples of lipids include LIPOFECTIN™,LIPOFECTAMINE™, DODAC/DOPE, and CHOL/DOPE. The resultingpolynucleotide-lipid complex can then be contacted with APCs.Alternatively, the polynucleotide can be introduced into APCs usingtechniques such as electroporation or calcium phosphate transfection.The polynucleotide-loaded APCs can then be used to stimulate cytotoxic Tlymphocyte (CTL) proliferation in vivo or ex vivo. In one embodiment,the ex vivo expanded CTL is administered to the subject in a method ofadoptive immunotherapy. The ability of the polynucleotide-loadedantigen-presenting cells to stimulate a CTL response can be determinedby known methods, for example by assaying the ability of effector cellsto lyse a target cell. Methods and compositions using antigen-presentingcells loaded with e.g., RNA are described in U.S. Pat. No. 6,306,388 toNair et al., which is incorporated herein by reference for its teachingof methods of generation and use of APCs loaded with RNA.

In another aspect, the present invention provides a compositioncomprising antigen-presenting cells that have been contacted in vitrowith at least one CMV antigen, or nucleic acids encoding the at leastone CMV antigen, under a condition sufficient for the at least one CMVantigen to be presented by the antigen-presenting cells.

In another aspect, the present invention provides a method for preparinglymphocytes specific for the at least one CMV antigen. The methodcomprises contacting lymphocytes with the antigen-presenting cellsdescribed above under conditions sufficient to produce CMVantigen-specific lymphocyte capable of eliciting an immune responseagainst a cell that expresses a CMV antigen. Thus, theantigen-presenting cells also can be used to provide lymphocytes,including T lymphocytes and B lymphocytes, for eliciting an immuneresponse against cell that expresses a CMV antigen.

In one embodiment, a preparation of T lymphocytes is contacted with theantigen-presenting cells described above for a period of time,preferably for at least about 24 hours, for priming the T lymphocytes tothe at least one CMV antigen presented by the antigen-presenting cells.

For example, in another embodiment, a population of antigen-presentingcells can be co-cultured with a heterogeneous population of peripheralblood T lymphocytes together with at least one CMV antigen, or nucleicacids comprising the at least one CMV antigen. The cells can beco-cultured for a period of time and under conditions sufficient for theCMV antigens or their processed forms to be presented by theantigen-presenting cells and the antigen-presenting cells to prime apopulation of T lymphocytes to respond to cells that express a CMVantigen. Accordingly, T lymphocytes and B lymphocytes that are primed torespond to cells that express a CMV antigen can be prepared.

As described herein, the ability to induce lymphocytes to exhibit animmune response can be determined by any method including, but notlimited to, determining T lymphocyte cytolytic activity in vitro usingfor example CMV antigen-specific antigen-presenting cells as targets ofCMV antigen-specific cytolytic T lymphocytes (CTL); assaying CMVantigen-specific T lymphocyte proliferation; and determining B cellresponse to cells expressing a CMV antigen using, for example, ELISAmethods.

T lymphocytes can be obtained from any suitable source such asperipheral blood, spleen, and lymph nodes. The T lymphocytes can be usedas crude preparations or as partially purified or substantially purifiedpreparations, which can be obtained by standard techniques including,but not limited to, methods involving immunomagnetic or flow cytometrytechniques using antibodies.

Also contemplated within the scope of the present invention are cellsthat have been modified genetically for specific recognition of a cellthat expresses a CMV antigen. (e.g., T-cells genetically engineered toexpress cell-specific antibodies on their surface). In some embodiments,antigen-specific T cells are modified by gene transfer techniques knownin the art to express one or more heterologous genes, for example amarker gene or a gene whose gene product can enhance or impart aparticular phenotype or function to the antigen-specific T cell. Thus,for example, a marker gene can be expressed within activated T cellsresponding to antigen pulsed dendritic cells and allow for the selectiveenrichment and modification of antigen-specific T cells. By way ofanother example, antigen-specific T cells can be modified to express areceptor (e.g., a chemokine receptor) to migrate toward a ligand of thereceptor in vitro and in vivo.

In other aspects, the present invention provides a compositioncomprising the antigen-presenting cells or the lymphocytes describedabove, and a pharmaceutically acceptable carrier and/or diluent. In someembodiments, the composition further comprises an adjuvant as describedabove.

In another aspect, the present invention provides a method for elicitingan immune response to the cell that expresses a CMV antigen, the methodcomprising administering to the subject the antigen-presenting cells orthe lymphocytes described above in effective amounts sufficient toelicit the immune response. In some embodiments, the invention providesa method for treatment or prophylaxis of a neoplastic disease orsymptoms associated with CMV, the method comprising administering to thesubject an effective amount of the antigen-presenting cells or thelymphocytes described above. In one embodiment, the antigen-presentingcells or the lymphocytes are administered systemically, preferably byinjection. Alternately, one can administer locally rather thansystemically, for example, via injection directly into tissue,preferably in a depot or sustained release formulation. Furthermore, onecan administer in a targeted drug delivery system, for example, in aliposome that is coated with tissue-specific antibody. The liposomes canbe targeted to and taken up selectively by the tissue. In anotherembodiment, the invention provides use of the antigen-presenting cellsor the lymphocytes in the preparation of a medicament for eliciting animmune response to a cell that expresses a CMV antigen, preferably fortreating or preventing cancer.

Accordingly, the antigen-primed antigen-presenting cells of the presentinvention and the antigen-specific T lymphocytes generated with theseantigen-presenting cells can be used as active compounds inimmunomodulating compositions for prophylactic or therapeuticapplications for cancer. In some embodiments, the CMV antigen-primedantigen-presenting cells of the invention can be used for generatingCD8+ CTL, CD4+ CTL, and/or B lymphocytes for adoptive transfer to thesubject. Thus, for example, CMV antigen-specific CTLs can be adoptivelytransferred for therapeutic purposes in subjects afflicted with amalignant tumor such as a glioma.

The antigen-presenting cells and/or lymphocytes described above can beadministered to a subject, either by themselves or in combination, foreliciting an immune response, particularly for eliciting an immuneresponse to cells that express a CMV antigen. Such cell-basedcompositions are useful, therefore, for treating or preventing cancer.The cells can be introduced into a subject by any mode that elicits thedesired immune response to cells that express a CMV antigen.Furthermore, the antigen-presenting cells and/or lymphocytes can bederived from the subject (i.e., autologous cells) or from a differentsubject that is MHC matched or mismatched with the subject (e.g.,allogeneic). The injection site can be selected from subcutaneous,intraperitoneal, intramuscular, intradermal, intravenous, orintralymphoid.

Single or multiple administrations of the antigen-presenting cells andlymphocytes can be carried out with cell numbers and treatment beingselected by the care provider (e.g., physician). Preferably, theantigen-presenting cells and/or lymphocytes are administered in apharmaceutically acceptable carrier. Suitable carriers can be the growthmedium in which the cells were grown, or any suitable buffering mediumsuch as phosphate buffered saline. The cells can be administered aloneor as an adjunct therapy in conjunction with other therapeutics.

Accordingly, the invention contemplates methods for treatment and/orprophylaxis of a cell that expresses a CMV antigen, preferably aCMV-associated cancer cell, the method comprising administering to asubject in need of such treatment or prophylaxis atherapeutically/prophylactically effective amount of a composition asdescribed herein.

Techniques for formulating and administering can be found in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latestedition. Suitable routes can, for example, include oral, rectal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections. For injection,the therapeutic/prophylactic compositions of the present invention canbe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiological saline buffer.

F. Antibodies

The compositions of the present invention also can be used to raiseantibodies targeting a cell that expresses a CMV antigen. Accordingly,in other aspects, the composition and methods of the present inventionprovide one or more antibodies against the cell that expresses a CMVantigen, which antibodies themselves have many uses such as, forexample, passive immunization or target-specific delivery for effectorsas well as uses for diagnostic tests and kits based upon immunologicalbinding. Thus, in some embodiments, the present invention providesCMV-associated, cancer cell-specific antibodies that can be used intherapeutic and/or diagnostic applications.

The antibodies of the present invention can be used in screening ordiagnostic applications. The antibodies according to the presentinvention are valuable for in vitro and in vivo diagnostic purposes. Forexample, the antibodies can be used in western blots,immunoprecipitation, enzyme-linked immunosorbent assay (ELISA),fluorescence activated cell sorting (FACS), indirect immunofluoresencemicroscopy, immunohistochemistry (IHC), etc. In one embodiment, thepresent invention provides an immunological method for determining acell that expresses a CMV antigen, the method comprising contacting thecell with at least one antibody as disclosed herein.

For example, the antibodies can be used as diagnostic agents forassaying for the detection of CMV antigen expressing cells. Theantibodies of the present invention should be particularly suitable asdiagnostic agents given their binding affinity to a cell that expressesa CMV antigen. Essentially, a sample comprising cells (e.g., cancercells) will be incubated with the antibodies for a sufficient time topermit immunological interactions to occur. Those skilled in the artwill recognize that there are many variations in these basic procedures.These variations include, for example, RIA, ELISA, precipitation,agglutination, complement fixation and immunofluorescence. Preferably,the subject antibodies will be labeled to permit the detection ofantibody-cell immunocomplexes. Further, the antibodies of the presentinvention are also useful for detection and quantification of cells thatexpress a CMV antigen in vitro, or to kill and eliminate such cells froma population of mixed cells as a step in the purification of othercells.

The labels that are used in making labeled versions of the antibodiesinclude moieties that may be detected directly, such as radiolabels andfluorochromes, as well as moieties, such as enzymes, that must bereacted or derivatized to be detected. Radiolabels include, but are notlimited to, ⁹⁹Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ^(113m)In, ⁹⁷Ru, ⁶²Cu,⁶⁴¹Cu, ⁵²Fe, ^(52m)Mn, ⁵¹Cr, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er,¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm,¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh,and ¹¹¹Ag. The radiolabel can be detected by any of the currentlyavailable counting procedures.

An enzyme label can be detected by any of the currently utilizedcalorimetric, spectrophotometric, fluorospectrophotometric or gasometrictechniques. The enzyme is combined with the antibody with bridgingmolecules such as carbodiimides, periodate, diisocyanates,glutaraldehyde and the like. Many enzymes which can be used in theseprocedures are known and can be utilized. Examples are perioxidase,alkaline phosphatase, β-glucuronidase, β-D-glucosidase, urease, glucoseoxidase plus peroxidase, galactose oxidase plus peroxidase and acidphosphatase. Fluorescent materials which may be used include, forexample, fluorescein and its derivatives, rhodamine and its derivatives,auramine, dansyl, umbelliferone, luciferia,2,3-dihydrophthalazinediones, horseradish peroxidase, alkalinephosphatase, lysozyme, and glucose-6-phosphate dehydrogenase. Theantibodies may be tagged with such labels by known methods. Forinstance, coupling agents such as aldehydes, carbodiimides, dimaleimide,imidates, succinimides, bis-diazotized benzadine and the like may beused to tag the antibodies with the above-described fluorescent,chemiluminescent, and enzyme labels. Various labeling techniques aredescribed in Morrison, Methods in Enzymology, (1974), 32B, 103; Syvanenet al., J. Biol. Chem., (1973), 284, 3762; and Bolton and Hunter,Biochem J., (1973), 133, 529.

The antibodies and labeled antibodies may be used in a variety ofimmunoimaging or immunoassay procedures to detect the presence of cellsthat express a CMV antigen in a subject or monitor the status of suchcells or cancer in the subject. When used to monitor the status of acell that expresses a CMV antigen, a quantitative immunoassay procedurecan be used. For example, if such monitoring assays are carried outperiodically and the results compared, a determination can be maderegarding whether a subject's tumor burden has increased or decreased,wherein the tumor is a CMV-associated tumor. Common assay techniquesthat may be used include direct and indirect assays.

For example, in the case of therapeutic applications, the antibodies canbe used to inhibit a target involved in disease progression or to bringabout the cytotoxic death of target cells. Also, such therapeuticantibodies can inhibit a signaling pathway or induce antibody-dependentcell-mediated cytotoxicty, complement-dependent cytotoxicty, etc.

The antibodies of this invention thus provide effective targetingmoieties that can, but need not, be transiently or permanently coupledto an effector (thereby forming a hybrid molecule or chimeric moiety)and used to direct that effector to a particular target cell (e.g., acancer cell).

The effector molecule refers to a molecule or group of molecules that isto be specifically transported to the target cell. The effector moleculetypically has a characteristic activity that is to be delivered to thetarget cell. Effector molecules include, but are not limited tocytotoxins, labels, radionuclides (e.g., ²¹¹At), ligands, antibodies,drugs, liposomes, epitope tags, and the like. Preferred effectorsinclude cytotoxins (e.g., Pseudomonas exotoxin, gelonin, ricin, abrin,Diphtheria toxin, and the like), immunomodulators as described herein,or cytotoxic drugs or prodrugs, in which case the hybrid molecule mayact as a potent cell-killing agent specifically targeting the cytotoxinto cells expressing the CMV antigen.

Further examples of effectors include, but are not limited to, granzyme,luciferase, vascular endothelial growth factor, β-lactamase, Tr-apo-1,Ang II, TAT, alkylating agents, daunomycin, adriamycin, chlorambucil,anti-metabolites (e.g., methotrexate), modaccin A chain, alpha-sarcin,Aleurites fordii proteins, dianthin proteins, Phytolacca americanaproteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor,curcin, crotin, Sapaonaria officinalis inhibitor, mitogellin,restrictocoin, phenomycin and enomycin.

In still other embodiments, the effector can include a liposomeencapsulating a drug (e.g., an anti-cancer drug such as doxirubicin,vinblastine, taxol, or other chemotherapeutic agents described herein),an antigen that stimulates recognition of the bound cell by componentsof the immune system, and antibody that specifically binds immune systemcomponents and directs them to the cells that express a CMV antigen, andthe like.

Other suitable effector molecules include pharmacological agents orencapsulation systems containing various pharmacological agents. Thus,the targeting molecule of the hybrid molecule may be attached directlyto a drug that is to be delivered directly to cells that express a CMVantigen. Such drugs are well known to those of skill in the art andinclude, but are not limited to, doxirubicin, vinblastine, genistein, anantisense molecule, the various other chemotherapeutic agents describedherein, and the like.

Alternatively, the effector molecule may be an encapsulation system,such as a viral capsid, a liposome, or micelle that contains atherapeutic composition such as a drug, a nucleic acid (e.g., anantisense nucleic acid), or another therapeutic moiety that ispreferably shielded from direct exposure to the circulatory system.Methods of preparing liposomes attached to antibodies are well known tothose of skill in the art. See, for example, U.S. Pat. No. 4,957,735;Connor, J. et al. (1985) Pharmacol. Ther., 28: 341-365.

G. Determining CMV Nucleic Acid

In other aspects, the present invention provides compositions andmethods for determining CMV nucleic acid in a subject, preferably CMVDNA in blood or other biological fluid, e.g., for determiningsubclinical viremia in a sample of blood obtained from the subject.Accordingly, in various embodiments, the compositions and methodsprovide effective diagnostic, monitoring, and prognostic tests/assaysthat complement various diagnostic and/or therapeutic procedures andtreatments including methods described herein such as, for example,prophylactic and/or therapeutic treating of a disease or conditionassociated with a precancerous cell, a cancer cell, or a cell-typepredisposed to developing cancer associated with CMV.

By way of example, an amplification method is provided that is reliablein determining CMV reactivation in human patients undergoing allogeneicbone marrow transplantation (aBMT), and in some cases preceded detectionof viral reactivation by as much as six weeks compared to currently usedclinically approved diagnostic CMV PCR assays. As also illustrated byexamples provided herein, CMV DNA from surgically resected GBM specimenscan be analyzed using PCR amplification, for example, across a variableregion of the gB gene in HCMV genome (region 82801-84180, NCBI GenBankaccession#NC_(—)001347) and subsequent DNA sequencing.

In one embodiment, the invention provides a method of determining CMVnucleic acid in a subject, the method comprising: (a) amplifying anucleic acid molecule from a biological sample to obtain an amplicon,wherein amplifying comprises contacting the nucleic acid molecule withat least one pair of primers comprising a first primer and a secondprimer, wherein the amplicon is about 1000 base pairs (bp) or less inlength; and (b) determining presence of the amplicon, wherein presenceof the amplicon is indicative of CMV nucleic acid in the subject.

In some embodiments, the amplicon is no greater than about 1000 bp inlength, illustratively, no greater than about 900 bp, no greater thanabout 800 bp, no greater than about 700 bp, no greater than about 600bp, no greater than about 500 bp, no greater than about 400 bp, nogreater than about 300 bp, no greater than 200 bp, no greater than about150 bp, no greater than about 100 bp, no greater than about 90 bp, nogreater than about 80 bp, no greater than about 70 bp, no greater thanabout 60 bp, no greater than about 50 bp, and no greater than about 40bp in length. In some embodiments, the amplicon is 200 base pairs (bp)or less in length.

Non-limiting examples of primer pairs and their corresponding targetgenes are shown in Tables 1 and 2.

TABLE 1 Human CMV primer pairs for determining CMV DNA Name (Genes-Amplicon Detection Primers) Primer Pair Sequences (5′ to 3′)¹ Size (bp)Rate gB-E1E2 5′-tcc aac acc cac agt acc cgt-3′(SEQ ID NO: 6) 268  51/2235′-cgg aaa cga tgg tgt agt tcg-3′(SEQ ID NO: 7) (22.9%) gB-i1i25′-cgc cgc ccg ccc cgc gcc cgc cgc ggc agc acc 144 164/223tgg ct-3′(SEQ ID NO: 8) (73.5%)5′-gta aac cac atc acc cgt gga-3′(SEQ ID NO: 9) gB-i3i45′-gcc gcg gca gca cct ggc t-3′(SEQ ID NO: 10) 122 152/2235′-aac cac atc acc cgt gga-3′(SEQ ID NO: 11) (68.1%) gB-5/65′-tac ccc tat cgc gtg tgt tc-3′(SEQ ID NO: 12) 254  65/2235′-ata gga ggc gcc acg tat tc-3′(SEQ ID NO: 13) (29.1%) gB-5/75′-tac ccc tat cgc gtg tgt tc-3′(SEQ ID NO: 14) 320  11/505′-cct cct ata acg cgg ctg ta-3′(SEQ ID NO: 15) (22%) gB-7B/85′-tcc gaa gcc gaa gac tcg ta-3′(SEQ ID NO: 16) 410  26/505′-gat gta acc gcg caa cgt gt-3′(SEQ ID NO: 17) (52%) gB-9/105′-ttt gga gaa aac gcc gac-3′(SEQ ID NO: 18) 748  10/505′-cgc gcg gca atc ggt ttg ttg to-3′ (20%) (SEQ ID NO: 19) gp64-1/25′-ccg caa cct ggt gcc cat gg-3′(SEQ ID NO: 20) 138   7/505′-cgt ttg ggt tgc gca gcg gg-3′(SEQ ID NO: 21) (14%) gpUL73-5′-ttc ggt cgg tca aca tcg taa g-3′ 516   6/50 1/2 (SEQ ID NO: 22) (12%)5′-cac cca cgt atg taa acc tta c-3′(SEQ ID NO: 23) IE1-5′-aga aag atg tcc tgg cag aac t-3′ 605  37/223 A443/A444(SEQ ID NO: 24) (16.6%)5′-cct cag gta caa tgt agt tct c-3′(SEQ ID NO: 25) IE1-5′-aga aag atg tcc tgg cag aac t-3′ 422  40/223 A445/A446(SEQ ID NO: 26) (17.9%)5′-cct cag gta caa tgt agt tct c-3′(SEQ ID NO: 27) IE-1/25′-cgt cct tga cac gat gga gt-3′ (SEQ ID NO: 28) 311  10/505′-att ctt cgg cca act ctg ga-3′(SEQ ID NO: 29) (20%) IE-3/45′-ccc tga taa tcc tga cga gg-3′(SEQ ID NO: 30) 201  12/505′-cat agt ctg cag gaa cgt cgt-3′(SEQ ID NO: 31) (24%) IEA1-5′-caa gcg gcc tct gat aac caa gc-3′ 421   9/50 P1/P2 (SEQ ID NO: 32)(18%) 5′-ctc ttc ctc tgg ggc aac ttc ctc-3′ (SEQ ID NO: 33) MIE-P1/P25′-ggg tgc tgt cct gct atg tct ta-3′(SEQ ID 370  29/50 NO: 34) (58%)5′-cat cac tct gct cac ttt ctt cc-3′(SEQ ID NO: 35) pp65-1/25′- cac ctg tca ccg ctg cta tat ttg c-3′ 400  10/50 (SEQ ID NO: 36)(20%) 5′- cac cac gca gcg gcc ctt gat ctt t-3′ (SEQ ID NO: 37) pp65-3/45′-gac aca aca ccg taa agc-3′(SEQ ID NO: 38) 278   8/505′-cag cgt tcg tgt ttc c-3′(SEQ ID NO: 39) (16%) pp65-11/125′-agc gcg tac aca tag atc ga-3′(SEQ ID NO: 40) 186  12/505′-gct gat ctt ggt atc gca gta c-3′(SEQ ID (24%) NO: 41) pp65-13/145′-agt ggt gca cgt tga tgc tg-3′(SEQ ID NO: 42) 232  15/505′-tcg ctg atc ttg gta tcg ca-3′(SEQ ID NO: 43) (30%) UL54-5′-cta cac ggt agc gac gag ac-3′(SEQ ID NO: 44) 501   8/50 S1/AS15′-atg ttt cta ggc tac tct gac tg-3′(SEQ ID (16%) NO: 45) UL89/935′-gat ccg acc cat tgt cta ag-3′(SEQ ID NO: 46) 152  10/50 (EcoRI5′-ggc agc tat cgt gac tgg ga-3′(SEQ ID NO: 47) (20%) Fragment D-P1/P2)UL144-1/2 5′-gcc tct gat aat gct cat ctg c-3′(SEQ ID NO: 48) 400  12/505′-ggc tag agt atg acg acc gct t-3′ (24%) (SEQ ID NO: 49) UL144-5/65′-tcg ttg ttt gtg atg ttg gac gcc-3′(SEQ ID 265  21/50 NO: 50) (42%)5′-tga agt gca act ggg caa tga gtg-3′(SEQ ID NO: 51) UL144-7/85′-cgt tgt ttg tga tgt tgg acg cct-3′(SEQ ID 264  23/50 NO: 52) (46%)5′-tga agt gca actg ggc aat gag tg-3′(SEQ ID NO: 53) UL144-5′-ttg ttt gtg atg ttg gac gcc tgg-3′(SEQ ID 262  18/50 9/10 NO: 54)(36%) 5′-tga agt gca act ggg caa tga gtg-3′(SEQ ID NO: 55) UL144-5′-atg gtt ctt agg tgc gca tac ggt-3′(SEQ ID 223  17/50 11/12 NO: 56)(34%) 5′-tga agt gca act ggg caa tga gtg-3′(SEQ ID NO: 57) UL144-5′-agg cta gag tat gac gac cgc ttt-3′(SEQ ID 225  19/50 13/14 NO: 58)(38%) 5′-acg gca cgt atg tat cgg gac ttt-3′(SEQ ID NO: 59) US7/85′-gga tcc gca tgg cat tca cgt atg t-3′ 406   2/50 (HindIII-X(SEQ ID NO: 60) (40%) Fragment- 5′-gaa ttc agt gga taa cct gcg gcg a-3′P1/P2) (SEQ ID NO: 61) US28-5′-agc gtg ccg tgt acg t ta c-3′(SEQ ID NO: 62) 412  14/50 10/115′-ata aag aca agc acg acc-3′(SEQ ID NO: 63) (28%) ¹Primers weredesigned in lab with NTI Advance 10 software (Invitrogen, Carlsbad, CA)

TABLE 2Quantitative PCR results on peripheral blood samples from patients undergoingallogeneic bone marrow transplantation. Lowest Ct Threshold Value to ofDetect HCMV Detection 10 Genes- Sequences of Primers & Probes¹ Detection(copy#/ copies of Primers 5′-3′ Rate qPCR) HCMV gB-18/19/205′-aaa gag ctg cgt tcc agc aa-3′ (SEQ ID  9/30 10 38.1 NO: 64) (30%)5′-gag gtc gtc cag acc ctt ga-3′ (SEQ ID NO: 65)5′-[FAM]-cat gcg cga att caa ctc gta caa gc-[TAMRA]-3′ (SEQ ID NO: 66)gB21/22/23 5′-atc gtg aga cct gta atc tga act gta-3′ 16/30 1 31.5(SEQ ID NO: 67) (53.3%) 5′-gga agt tgc aaaa aaa tga taag ga-3′(SEQ ID NO: 68) 5′-[FAM]-tga cca tca cta ctg cgc gct cca- [TAMRA]-3′(SEQ ID NO: 69) Pp65- 5′-ggc tac ggt tca ggg tca ga-3′ (SEQ ID 9 /30 1037.4 1wx/2wx/3wx NO: 70) (30%) 5′-ccg ggc aag gcg tctt-3′ (SEQ IDNO: 71) 5′-[FAM]-tgg gac gcc aac gac atc tac cg- [TAMRA]-3′(SEQ ID NO: 72) Pp65- 5′-gcg cac gag ctg gtt tg-3′ (SEQ ID  9/30 10 34.54wx/5wx/6wx NO: 73) (30%) 5′-aca cct tga cgt act ggt cac cta t-3′(SEQ ID NO: 74) 5′-[FAM]-acg cgc gca acc aag atg cag- [TAMRA]-3′(SEQ ID NO: 75) Pp65-7/8/9 5′-aca cat aga tcg aca tgg gct cct-3′ (SEQ9 /30 10 35.1 ID NO: 76) (30%) 5′-tgc agg tgc agc aca cgt act tta-3′(SEQ ID NO: 77) 5′-[FAM]-ttg tgc acg ttg acc gac acg ttc t-[TAMRA]-3′(SEQ ID NO: 78) Pp-65- 5′-tcg cgc ccg aag agg-3′ (SEQ ID  9/30 10 35.321/22/23 NO: 79) (30%) 5′-cgg ccg gat tgt gga tt-3′ (SEQ ID NO: 80)5-[FAM]-cac cga cga gga ttc cga caa cg- [TAMRA]-3′ (SEQ ID NO: 81) Pp65-5′-gca gcc acg gga tcg tac t-3′ (SEQ ID 11/30 2 33.9 24/25/26 NO: 82)(36.7%) 5′-ggc ttt tac ctc aca cga gca tt-3′ (SEQ ID NO: 83)5′[FAM]-cgc gag acc gtg gaa ctg cg- [TAMRA]-3′ (SEQ ID NO: 84) Pp65-5′-gtc agc gtt cgt gtt tcc ca-3′ (SEQ ID  9/30 10 35.7 27/28/29 NO: 85)(30%) 5′-ggg aca caa cac cgt aaa gc-3′ (SEQ ID NO: 86)5′-[FAM-5′-ccc gca acc cgc aac cct tca tg-[TAMRA]-3′ (SEQ ID NO: 87)Pp65- 5′-gcg gta aga cgg gca aat ac-3′ (SEQ ID  9/30 10 35.2 30/31/32NO: 88) (30%) 5′-ggc gtc gag atg ttc gta gag-3′ (SEQ ID NO: 89)5′-[FAM]-cac cat cga cac cac acc ctc atg a-[TAMRA]-3′ (SEQ ID NO: 90)US28-1/2/3 5′-cag cgt gcc gtg tac gtt act-3′ (SEQ ID 10/30 5 33.1NO: 91) (33.3%) 5′-gtg caa tct ccg tga taa aac aca-3′ (SEQ ID NO: 92)5′-[FAM]-act gcc tgt ttc tac gtg gct atg ttt gcc-[TAMRA]-3′(SEQ ID NO: 93) US28-4/5/6 5′-tgg cta tgt ttg cca gtt tgt g-3′ (SEQ ID 9/30 10 37.3 NO: 94) (30 5′-cag gcc gat atc tca tgt aaa caa t-3′(SEQ ID NO: 95) 5′-[FAM]-ttt atc acg gag att gca ctc gatcgc t-[TAMRA]-3′ (SEQ ID NO: 96) US28-7/8/95′-gat gca ata cct cct aga tca caa ctc-3′ 11/30 2 32.0 (SEQ ID NO: 97)(36%) 5′-gca aac ata gcc acg tag aaa ca-3′ (SEQ 7%) ID NO: 98)5′-[FAM]-cca gcg tgc cgt gta cgt tac tca ctg-[TAMRA]-3′ (SEQ ID NO: 99)HXFL4- 5′-aag cgc tgg ata cac ggt aca-3′ (SEQ 11/30 2 32.2 1/2/3ID NO: 100) (36.7%) 5′-gaa tac aga cac tta gag ctc ggg gt-3′(SEQ ID NO: 101) 5′-[FAM]-ctg gcc agc acg tat ccc aac agc a-[TAMRA]-3′(SEQ ID NO: 102) IE-5/6/7 5′-caa gaa ctc agc ctt ccc taa gac-3′ (SEQ 9/30 10 38.0 ID NO: 103) (30%) 5′-tga ggc aag ttc tgc aat gc-3′ (SEQ IDNO: 104) 5′-[FAM]-cca atg gct gca gtc agg cca tg- [TAMRA]-3′(SEQ ID NO: 105) IE-8/9/10 5′-cag att aag gtt cga gtg gac atg-3′ 11/30 233.9 (SEQ ID NO: 106) (36.7%) 5′-agg cgc cag tga att tct ctt-3′ (SEQ IDNO: 107) 5′-[FAM]-tgc ggc ata gaa tca agg agc aca tg-[Tamra]-3′(SEQ ID NO: 108) ¹Probes are shown as dual-labeled (5′ FAM/3′ TAMRA)with both a fluorophore and a quencher dye as used in real-time PCR.

In one embodiment, the first primer and the second primer are sufficientto provide for an amplicon that corresponds to a region of the gB (e.g.,UL55) or MIE gene. However, any primer pair that can provide an ampliconcorresponding to any region of a CMV nucleic acid also is within thescope of the present invention. In some embodiments, a primer for use inaccordance with the present invention comprises a nucleotide sequence asshown in SEQ ID Nos:8-63.

In another embodiment, the first primer and the second primer correspondto a primer pair, wherein the first primer comprises a first primersequence as shown by SEQ ID NO: 8, wherein the second primer comprises asecond primer sequence as shown by SEQ ID NO:9. In other embodiments,the first primer and the second primer correspond to a primer pair,wherein the first primer comprises a first primer sequence as shown bySEQ ID NO:10, wherein the second primer comprises a second primersequence as shown by SEQ ID NO:11.

In another embodiment, the first primer and the second primer correspondto a primer pair, wherein the first primer comprises a first primersequence as shown by SEQ ID NO:16, wherein the second primer comprises asecond primer sequence as shown by SEQ ID NO:17. In one embodiment, thefirst primer and the second primer correspond to a primer pair, whereinthe first primer comprises a first primer sequence as shown by SEQ IDNO:34, wherein the second primer comprises a second primer sequence asshown by SEQ ID NO:35.

In other embodiments, the invention provides a method of determining CMVnucleic acid in a subject, the method comprising: (a) amplifying anucleic acid molecule from a biological sample to obtain an amplicon,wherein amplifying comprises contacting the nucleic acid molecule withat least one pair of primers and at least one corresponding probe asshown in Table 2. In one embodiment, the pair of primers have a sequenceshown as SEQ ID NOs:67 and 68; and the corresponding probe has asequence shown as SEQ ID No:69.

In some embodiments, a method of determining CMV nucleic acid in thesubject further comprises subjecting the biological sample tosnap-freezing. Snap-freezing methods are well known in the art and canbe performed using a variety of reagents and techniques including, forexample, snap-freezing in liquid nitrogen or alcohol/dry ice (e.g.,MeOH/dry ice).

Preferably, a method further comprises concentrating the nucleic acidfollowing the step of snap-freezing. In one embodiment, the step ofconcentrating comprises precipitating the nucleic acid following thestep of snap-freezing. In another embodiment, precipitating comprisescontacting the nucleic acid with an alcohol (e.g., EtOH precipitation).

In still further embodiments, a step of determining the presence of anamplicon is performed by any suitable method of detection of theamplicon, for example a detection method that is characterized as havinga sensitivity of detection of at least 10-fold that of an agarose gelstained with ethidium bromide.

In other aspects, the present invention provides a method of elicitingin a subject an immune response to a cell, the method comprising:

(a) amplifying a nucleic acid molecule from a biological sample from thesubject to obtain an amplicon, wherein amplifying comprises contactingthe nucleic acid molecule with at least one pair of primers comprising afirst primer and a second primer, wherein the amplicon is about 1000base pairs (bp) or less in length;

(b) determining presence of the amplicon, wherein presence of theamplicon is indicative of CMV nucleic acid in the subject; and

(c) administering to the subject a pharmaceutically acceptablecomposition comprising at least one CMV antigen, or nucleic acidsencoding the at least one CMV antigen, wherein the pharmaceuticallyacceptable composition, when administered to the subject, elicits animmune response to the cell. The steps (a), (b), and (c) are asdescribed above.

In another aspect, the present invention provides a method of elicitingin a subject an immune response to a cell, the method comprising:

(a) amplifying a nucleic acid molecule from a biological sample from thesubject to obtain an amplicon, wherein amplifying comprises contactingthe nucleic acid molecule with at least one pair of primers comprising afirst primer and a second primer, wherein the amplicon is about 1000base pairs (bp) or less in length;

(b) determining presence of the amplicon, wherein presence of theamplicon is indicative of CMV nucleic acid in the subject; and

(c) administering to the subject a composition comprising an effectiveamount of antigen presenting cells, T-lymphocytes, or both, wherein theantigen presenting cells and T lymphocytes have been sensitized in vitrowith a sensitizing-effective amount of at least one CMV antigen, whereinthe effective amount of antigen presenting cells, T lymphocytes, or bothis sufficient to elicit the immune response to the cell that expressesthe CMV antigen.

In one aspect, the present invention provides a method of reducing orinhibiting growth or spread of a cell that expresses a CMV antigen, themethod comprising:

(a) amplifying a nucleic acid molecule from a biological sample from thesubject to obtain an amplicon, wherein amplifying comprises contactingthe nucleic acid molecule with at least one pair of primers comprising afirst primer and a second primer, wherein the amplicon is about 1000base pairs (bp) or less in length;

(b) determining presence of the amplicon, wherein presence of theamplicon is indicative of CMV nucleic acid in the subject; and

(c) administering to a subject a therapeutically or prophylacticallyeffective amount of a pharmaceutically acceptable composition to reduceor inhibit growth or spread of the cell in the subject. The steps (a),(b), and (c) are as described above.

In still further aspects, the present invention provides a method formonitoring the therapeutic efficacy of treating a subject having cellsthat expresses a CMV antigen, the method comprising:

(a) determining, in a first biological sample from the subject, a firstamount of CMV nucleic acid, wherein determining comprises: amplifying anucleic acid molecule from the biological sample to obtain an amplicon,wherein amplifying comprises contacting the nucleic acid molecule withat least one pair of primers comprising a first primer and a secondprimer, wherein the amplicon is about 1000 base pairs (bp) or less inlength, wherein presence of the amplicon is indicative of CMV nucleicacid in the subject;

(b) treating the subject;

(c) determining, after a suitable period of time, a second amount of CMVnucleic acid in a second sample obtained from the treated subject; and

(d) comparing the first amount with the second amount, wherein adifference between the first amount and the second amount is indicativeof effectiveness of the treatment.

In some embodiments, the treating in step (b) comprises administering tothe subject a therapeutic or prophylactic amount of a pharmaceuticalcomposition as described above.

In other embodiments, the present invention provides assays directed todetermining a pharmaceutical agent having activity against a cell thatexpresses a CMV antigen, the method comprising:

(a) determining, in a first biological sample from the subject, a firstamount of CMV nucleic acid, wherein determining comprises: amplifying anucleic acid molecule from the biological sample to obtain an amplicon,wherein amplifying comprises contacting the nucleic acid molecule withat least one pair of primers comprising a first primer and a secondprimer, wherein the amplicon is about 1000 base pairs (bp) or less inlength, wherein presence of the amplicon is indicative of CMV nucleicacid in the subject;

(b) treating the subject with the pharmaceutical agent;

(c) determining, after a suitable period of time, a second amount of CMVnucleic acid in a second sample obtained from the treated subject; and

(d) comparing the first amount with the second amount, wherein thepharmaceutical agent has activity against the cell when the secondamount is less than the first amount.

III. Kits

The compositions of the present invention can be supplied in unit dosageor kit form. Kits can comprise various components of thepharmaceutically acceptable composition or vaccines thereof provided inseparate containers as well as various other active ingredients oragents including chemotherapeutic agents. For example, the containerscan separately comprise at least one CMV antigen or nucleic acidsencoding the at least one CMV antigen such that when combined with othercomponents of the kit together constitute a pharmaceutically acceptablecomposition in unit dosage or multiple dosage form. Preferred kits atleast comprise, in separate containers, a source of antigens (e.g., theat least one CMV antigen or nucleic acids encoding them); and one ormore adjuvants (e.g., cytokines). The kit can further comprise aphysiologically acceptable carrier, diluent, or excipient in a separatecontainer. Optionally, the kit can further comprise a delivery agentsuch as nanoparticles or transfection reagents. Packaged compositionsand kits of this invention also can include instructions for storage,preparation, and administering. One or more nucleic acid primers inaccordance with the present invention are optionally included in thekit, preferably provided in one or more separate containers.

The present invention will be illustrated in more detail by way ofExamples, but it is to be noted that the invention is not limited to theExamples.

EXAMPLES Example 1 HCMV Proteins are Expressed in Malignant Gliomas

To determine whether HCMV proteins were expressed in malignant gliomas(MGs), paraffin sections from 45 GBM specimens selected from our braintumor bank were examined by immunohistochemistry (IHC).

Human GBM, oligodendroglioma, meningioma, ependymoma, and normal brainsurgical specimens were obtained in paraffin blocks (with InstitutionalReview Board [IRB] approval). Tumor specimens were requested based ondiagnosis only from the Preston Robert Tisch Brain Tumor Center at DukeTissue Bank. A total of 45 GBM cases confirmed by a neuropathologistwere selected (36 primary GBM and 9 recurrent GBM). The group consistedof 26 males and 19 females, with a median age of 51 years. Specimenswere sectioned (6 μm) and were blocked for endogenous peroxidase (3%H₂O₂, for 12 min) and incubated with Fc receptor blocker (10 min at 20°C.; Innovex Biosciences, Richmond, Calif., USA) before the addition of amonoclonal antibody (mAb). IHC was performed using three-stagehorseradish peroxidase detection systems (BioGenex, San Ramon, Calif.,USA; Dako, Carpinteria, Calif., USA; and Innovex Biosciences) with thefollowing mAbs: anti-IE1-72 (1:25; BioGenex), anti-pp65 (1:30;Novocastra, Newcastle upon Tyne, UK), and antismooth muscle actin (1:15;BioGenex). Antibody parameters (e.g., postfixation, retrieval, andincubation time) were established for each mAb using DAB (InnovexBiosciences) as chromogen. Primary glioma cultures established for 14 to21 days from freshly resected GBM specimens were fixed and permeabilizedusing cold methanol, followed by postfixation for 10 min with 10%neutral buffered formalin. Blocking of nonspecific binding was conductedusing biotin block and avidin block (BioGenex) and FC receptor blockade(Innovex). Incubation with primary antibodies using isotype controls(mouse IgG1, mouse IgG2a; Invitrogen, Carlsbad, Calif., USA), CD45antibody (BD Biosciences, San Jose, Calif., USA), pp28 antibody(Virusys, Sykesvile, Md., USA), glycoprotein B (gB; Virusys), and HIVp17 (Virogen, Watertown, Mass., USA) was conducted for 2 h or overnightat 4° C. (1 μg/ml antibody concentration) and detection conducted usingBioGenex three-stage horseradish detection system.

Detection was conducted using a mAb specific for the HCMV-encodedantigen, IE1-72. IE1-72 immunoreactivity was detected in 42 out of 45(93%) specimens examined by IHC. Strong nuclear and cytoplasmic stainingwas detected in tumor cells and occasionally endothelial cells as well(FIG. 1). However, infiltrating lymphocytes and surrounding normal brainareas were devoid of immunoreactivity to the IE1-72 antibody. To furtherconfirm specific detection of HCMV, 33 of the 45 cases were examined forreactivity to a mAb specific for the HCMV matrix protein, pp65. Thirtyof the 33 cases (91%) were immunoreactive for pp65 in the tumor cellsbut not in areas of adjacent normal brain. pp65 reactivity was ingeneral less ubiquitous than IE1-72 detection in tumor cells, but amajority of tumor cells in all specimens examined displayedimmunoreactivity against the pp65 antibody (FIG. 1; Table 3).

TABLE 3 Summary of HCMV detection in GBM specimens HCMV GBM TissueSpecimen Primary GBM Cultures IE1 IHC 42/45 (93%)^(a)  4/4 (100%) pp65IHC 30/33 (91%)^(a) 12/12 (100%) HCMV DNA ISH 16/16 (selected cases) nottested gB PCR 21/34 (61.7%)^(b)  13/17 (70.6%) IE1 PCR  8/34 (24%)^(b)9/17 (53%) Abbreviations: HCMV, human cytomegalovirus; GBM, glioblastomamultiforme; IHC, immunohistochemistry; ISH, in situ hybridization; gB,glycoprotein B; PCR, polymerase chain reaction. ^(a)Other tumors testedby IHC were negative for HCMV: oligodendroglioma (n 5 = 5); one caseexhibited focal detection of HCMV IE1 in endothelial cells but noreactivity within tumor parenchyma); meningioma (n = 5); ependymoma (n =5). ^(b)PCR products were isolated from 21 gB PCR reactions and 6 IE1PCR and confirmed by DNA sequencing to be specific for HCMV.

To rule out the possibility of nonspecific detection in tumor cells, IHCwas performed on tumor sections with isotype- and concentration-matchedcontrol mAbs. Isotype-matched, control antibodies used at identicalconcentration to the HCMV-specific mAbs showed no immunoreactivitywithin tumor cells, and an isotype-matched mAb to smooth muscle actindemonstrated reactivity to blood vessels within tumor and normal brainsections (FIG. 1A) but no reactivity with tumor cells. Examinationresults of meningiomas (n=5), ependymomas (n=5), and oligodendrogliomas(n=5) were negative for detection of IE1 and pp65, except for focalendothelial staining observed in a single case of oligodendroglioma withthe IE1 monoclonal antibody (Table 3).

Example 2 PCR Detection of HCMV in Malignant Glioma Specimens

As shown in FIG. 2, PCR amplification of gB in 11/13 high grade MGs (9/11 GBMS, 2/2 AAs) was performed. Forty cycles of PCR were conductedusing gB specific primers to amplify a 122 bp product. HCMV gB wassequence verified in 17 of 32 positive samples.

Example 3 CMV Antigens are Present in Pre-Malignant Lesions

The presence of CMV IE1 and pp65 proteins in greater than about 75% ofpremalignant colorectal polyps and greater than about 80% ofadenocarcinomas of the colon but not in adjacent non-neoplastic colonbiopsy samples are shown (See, e.g., Harkins et al., Lancet, 360:1557(2002)). Furthermore, the presence of CMV in about 48% of low gradegliomas, which frequently progress to high grade lesions, is shown (See,e.g., Scheurer et al., Detection of Human Cytomegalovirus in DifferentHistological Types of Gliomas, Acta Neuropathologica, Epublished Mar.20, 2008, Springer Berlin/Heidelberg). Thus, the compositions andmethods of the present invention also can provide for preventing ortreating pre-neoplastic, low-grade, and/or pre-malignant lesions as wellas progression to neoplastic disease.

Example 4 Ex Vivo Generation of Autologous DC

Contents of a Leukapheresis Product (LP) bag was placed in a 1 L sterilecorning bottle and an equal volume of phosphate buffered saline (PBS)was added to dilute the LP. Using centrifuge tubes, 20 mL of Histopaque™(Sigma #1077-1) was gently over layered with 30 mL of the diluted LP,then spun at 1300×g for 25 minutes. The interface (1 interface per tube)was removed; PBS was added to 50 mL; and cells pelleted for 5 minutes at500×g room temperature (RT). The supernatant was decanted and the pelletwas resuspended in 50 mL PBS, then pelleted as above. The supernatantwas decanted and 2 pellets were combined in 50 mL PBS, then pelleted asabove. Again, the supernatant was decanted and 2 pellets were combinedin 50 mL PBS, then pelleted as above. Pellets were combined and washedwith PBS until all of the cells were combined into 1 tube.

Trypan blue exclusion was performed to determine the number of live anddead cells via with the aid of a hematocytometer. The cells wereresuspended in AIM V media (Life Technologies #870112dk) with 2% HumanAB Sera (HABS) (Valley Biomedical #HP1022) at 2×10⁸ per mL. Twenty-ninemL of AIM V media containing 2% HABS and 1 mL of PBMC cell suspensionwere added to a T-150 cell culture flask. All cell culture flasks wereplaced into a single dedicated humidified incubator at 37° C., 5% CO₂for 2 hours to allow the monocyte precursors to adhere. Following theadherence period, non-adherent cells were removed and the remainingmonolayer was washed once with PBS. The adherent cells were replenishedwith 30 ml of AIM-V per flask supplemented with 800 U/mL recombinanthuman GM-CSF (Berlex Laboratories, Inc.) and 500 U/mL recombinant humanIL-4 (R&D Systems #204-IL/CF), and incubated in humidified incubator at37° C., 5% CO₂ for 7 days.

After the 7-day culture period, adherent DCs were washed with cold PBS.Ten ml of Dissociation Buffer Enzyme-Free (Life Technologies #13150-016)was added and the DCs were incubate at 4° C. for 10 minutes. Theremainder of adherent DC were flushed from the flask and combined withpreviously harvested DC, then pelleted at 500×g for 10 minutes at 4° C.Following one wash with cold PBS, the cells were checked for number andviability.

Example 5 RNA Loading & Maturation of Dendritic Cells

Dendritic cells generated as described above were resuspended at 2.5×10⁷cells per mL of ViaSpan (Belzer UW-CSS, DuPont Pharmaceuticals,Wilmington, Del.). Two hundred μL of the suspension was then placed in acuvette (Gene Pulser Cuvette, Bio-Rad #165-2086) along with 10 μg ofpp65 RNA and the cells were electroporated (BTX Electro Square Porator#ECM830) at 300 volts for 500μ seconds. Approximately 1×10⁸ of theelectroporated cells were transferred to a T225 flask containing 50 mLof AIM V supplemented with 800 U/mL of recombinant human GM-CSF (BerlexLaboratories, Inc.) and 500 U/ml recombinant human IL-4 (R&D Systems#204-IL/CF), then incubated at 37° C., 5% CO₂ for 1 hour. At the end ofthe incubation period, the appropriate amount of media with maturationcytokine was added to a final concentration of the cytokines in thematuration cocktail of 10 ng/ml TNF-α (R&D Systems, #210-TA/CF); 10ng/ml IL-113 (R&D Systems, #201-LB/CF); and 1000 units/ml IL-6 (R&DSystems, #208-IL/CF). The cells were incubated overnight at 37° C. and5% CO₂.

At the end of the maturation period, the supernatant was removed andplaced in chilled 50 ml conical tube on ice. The remaining monolayer waswashed with ice cold PBS; combined with supernatant; then pelleted at500×g for 5 minutes 4° C. The pellet was resuspended in 10 ml of icecold PBS and kept on ice. Ten to 20 ml of Dissociation BufferEnzyme-Free (Life Technologies #13150-016) was added and the cells keptat 4° C., and the progress of the cells coming off was monitored every 5minutes. The flask was washed twice with 10 ml of ice cold PBS, combinedwith the cells from the dissociation buffer, and pelleted using 500×g.The cells were combined with the other cells, and brought up to 50 mland counted.

Example 6 Preparation of Dendritic Cells for Vaccination

Mature antigen-loaded dendritic cells in a 50 mL conical centrifuge tubeand 25 ml of PBS was slowly add, then the cells were pelleted at 200×gfor 5 minutes at 22° C. The cells were resuspended in 10 ml of PBS and,using a 2 ml pipette, a 100 μl sample of the suspension was removed andthe cells counted using a hemocytometer and trypan blue as describedabove. Cells were determined to be ≧70% viable before proceeding. Cellswere pelleted at 500×g for 5 min at 22° C. and resuspended at 5×10⁴cells/mL in 0.9% sodium chloride. The cells were loaded into a 1 ccsyringe with 25 G ⅝ gauge needle. Samples were sent for Gram stain andendotoxin testing prior to administration.

Example 7 Vaccinating Subjects with Newly-Diagnosed GBMs Using CMVpp65-LAMP RNA-Loaded DCs

Twenty-five patients (with ≧3 CMV seropositive patients in eachrandomized group) with newly diagnosed GBM are enrolled within 6 weeksof resection. Only 1 dose level of DCs (2×10⁷) and 1 dose level of ALT(3×10⁷/Kg) is assessed. The decision to dose escalate in subsequenttrials is dependent on analysis of the safety and immunologic responsesobtained in this trial. Patients are followed until death. The study ishalted if any 2 patients in either group experience a drug-related GradeIV or irreversible Grade III toxicity. All patients undergo aleukapheresis after resection for harvest of PBLs for ALT and generationof DCs. FIG. 3 schematically illustrates the vaccination protocol.

Patients receive radiation therapy (RT) and concurrent TMZ at a standardtargeted dose of 75 mg/m²/d. Patients with progressive disease duringradiation and that are dependent on steroid supplements abovephysiologic levels at time of vaccination, unable to tolerate TMZ, andwhose DCs or PBLs fail to meet release criteria, are replaced. Remainingpatients then receive the initial cycle of TMZ at a standard targeteddose of 200 mg/m²/d for 5 days 3±1 weeks after completing RT and arerandomized to receive ALT or not simultaneous with DC vaccine #1. DCsare given intradermally and divided equally to both inguinal regions.Vaccine #2 and #3 occurs at 2 week intervals. All patients undergoleukapheresis again for immunologic monitoring with specific assessmentof baseline antigen-specific cellular and humoral immune responses andfurther DC generations 4±2 weeks after vaccine #3. Patients are thenvaccinated monthly in conjunction with subsequent TMZ cycles every 5±1weeks for a total of 6 cycles after RT. TMZ is given on days 1-5 withDCs given on day 21±7 days. DC vaccinations continue after TMZ cyclesare finished.

Patients are imaged bimonthly without receiving any other prescribedanti-tumor therapy and continue with vaccinations until progression.Patients undergo an additional leukapheresis for generation of DCs ifneeded to continue vaccinations. At vaccine #4, patients are randomizedto different skin preparations at the inguinal sites and receive¹¹¹In-labeled DCs to compare the effects of different skin preparationson DC migration. When progression occurs, patients receive finalintradermal vaccinations bilaterally in the inguinal region and at theangle of the jaw with ¹¹¹In-labeled DCs to compare the effects ofdifferent vaccination sites on DC migration. As part of standard carefor these patients, upon tumor progression, participants may undergostereotactic biopsy or resection. As this is not a research procedureconsent is obtained separately. However, if tissue is obtained, it isused to confirm tumor progression histologically and to assessimmunologic cell infiltration and pp65 antigen escape at the tumor site.

Study inclusion criteria include: age >18 years of age; GBM (WHO GradeIV) with definitive resection <6 weeks prior to enrollment, withresidual radiographic contrast enhancement on post-resection CT or MRIof <1 cm in maximal diameter in any axial plane; and KarnofskyPerformance Status (KPS) of >80% and a Curran Group status of I-IV.

Study exclusion criteria include radiographic or cytologic evidence ofleptomeningeal or multicentric disease at any time prior to vaccination;prior conventional anti-tumor therapy other than steroids, RT, or TMZ;pregnant or need to breast feed during the study period (Negative β-HCGtest required); requirement for continuous corticosteroids abovephysiologic levels at time of first vaccination; active infectionrequiring treatment or an 101.5° unexplained febrile (>101.5° F.)illness; known immunosuppressive disease or human immunodeficiency virusinfection; patients with unstable or severe intercurrent medicalconditions such as severe heart or lung disease; allergic or unable totolerate TMZ for reasons other than lymphopenia; or patients withprevious inguinal lymph node dissection.

Exemplary procedures that are investigational include: leukapheresis forthe generation on DCs and ALT; intradermal immunizations with antigenloaded DCs; ALT; intradermal immunization with ¹¹¹In labeled antigenloaded DCs and SPECT scans; and DTH testing with standard recallantigens and antigen loaded and naïve DCs.

Activities associated with the protocol that are considered standardcare activities include: external beam radiotherapy; Temozolomide (TMZ);MRI; and biopsy at recurrence.

Patients receive their own lymphocytes intravenously (through a catheterplaced in your vein) after pre-medication with Benadryl 25-50 mg and 650mg of Tylenol to prevent infusion reactions. Transfusion is receivedover 45 to 90 minutes depending upon weight and the number of cellsreceived. The probability of risk of infection is relatively low, giventhe small injection volume (1 mL divided between >2 intradermallocations) and the fact that the DCs are strictly tested for sterilityprior to each injection. The risk of infection due to potentialcontamination of the DCs in the laboratory is minimized by biosafetyquality assurance and testing. All cell cultures are handled understerile conditions in a core tissue culture facility dedicated to theprocessing of human cells. Prior to injection into patients, DCs passsterility tests in thighlycolate broth, tryptic soy blood agar, andinhibitory Sabouraud agar. Following injections, patients are monitoredthroughout the course of the study for any signs and symptoms ofinfection. If an active infection is suspected, patients are culturedand treated with appropriate antibiotics.

Patients' DCs are radiolabeled with ¹¹¹In for correlative studies. Theradiation exposure to the patient and health care personnel is minimalat the proposed doses, and is roughly equivalent to living in a highaltitude city such as Denver for 13 days, or taking 4 airplane flightsfrom New Your to Los Angeles. Therefore, no specific radiationprecautions are taken. SPECT images are obtained for analysis.

During the MRI, patients are given a contrast agent. The agent is givenroutinely to obtain enhanced MRI scans of the brain. The agent isadministered through the vein and requires the placement of an IVcatheter. The catheter placement is similar to drawing blood except thatthe catheter remains in the vein during the time the agent is activelydelivered.

Example 8 Time to Tumor Progression (TTP) is Increased for Patients withNewly-Diagnosed GBM Treated with CMV pp65 RNA Loaded DCs

DCs were generated from a leukapheresis in vitro by 7-day culture withGM-CSF and IL-4. For in vitro generation of DCs, PBMCs were obtained byleukapheresis and transported to a cell processing facility. Forpatients without sufficient venous access for leukapheresis a temporaryintravenous catheter was inserted.

At the end of the 7 day incubation for generating DCs a sample of themedia was taken for mycoplasma testing, and the cells were thenharvested and electroporated with pp65-LAMP mRNA (2 micrograms RNA permillion DCs). The DCs were placed in a flask with AIM V media withGM-CSF+IL-4+TNF-α+IL-6+IL-1β at 37° C., 5% CO₂ for 18-20 hours formaturation. The cells are washed twice with PBS and frozen at 5×10⁶cells/mL in 90% autologous human AB serum (Valley Biomedical,Winchester, Va. 22602), 10% DMSO and 5% glucose in a controlled-ratefreezer at a rate of 1° C./minute. The DCs were then stored until neededat −135° C. After freezing, an aliquot of cells was thawed and sent foraerobic and anaerobic bacterial cultures (1×10⁶ DCs) and fungal cultures(1×10⁶ DCs).

For each vaccination, DCs were rapidly thawed at 37° C., washed threetimes with PBS, assessed for viability, and counted. To proceed, a cellviability of ≧70% was obtained. The cell concentration was adjusted to4×10⁷ cells/mL and DCs were resuspended in preservative free saline andplaced into a sterile tuberculin syringe with a 27 gauge needle.

For all DC preparations, a sample of cells was sent for Gram stain andendotoxin testing prior to administration. DC vaccination was not givenuntil endotoxin testing passed (<5.0 E.U./Kg) and the Gram stain wasnegative. An aliquot of cells also was sent for aerobic and anaerobicbacterial cultures (1×10⁶ DCs) and fungal cultures (1×10⁶ DCs).

A Phase I/II clinical trial using autologous pp65 RNA loaded DCs wasinitiated. This trial has enrolled 21 patients with newly diagnosed GBMwho underwent gross total resection (>95%) followed by standard externalbeam radiation (60 Gy) and concurrent TMZ (75 mg/m²/d) for six weeksfollowed by monthly 5 day TMZ (150-200 mg/m²/d) for six cycles. Fivepatients progressed during radiation and were not treated on protocol.Two patients were treated off-protocol on a compassionate-use basis.Leukapheresis harvested post surgical resection and prior to initiationof TMZ was used to generate DCs and pp65 RNA electroporated autologousDCs (2×10⁷ DCs i.d.) were administered every two weeks for first threedoses after first TMZ cycle and monthly thereafter on day 21 of eachcycle. Patients were monitored by MRI (every two months) for tumorprogression and blood collected monthly for immunologic monitoring.Controls are age, prognostic factor matched with identical eligibilitycriteria to patients enrolled on ATTAC trial derived from MD Andersondatabase.

The results are shown in FIG. 4. At follow-up of 21 months, 13 of 16patients remained alive (range 7.6 months to 22 months post surgery)with a median TTP of 12.5 months and median overall survival has notbeen reached but is greater than 20.1 months, which is highly favorablecompared to historical controls (see, e.g., Stupp et al., N. Engl. J.Med. 352:987 (2005); Phase III TMZ/XRT+adjuvant TMZ median TTP is 7.1months and median survival 14.6 months).

Example 9 Overall Survival is Increased for GMB Patients Treated withCMV pp65 RNA-Loaded DC Vaccine

Following completion of RT with concurrent daily TMZ (75 mg/m²/d),patients were randomized to those that received DC vaccinations only andthose that received ALT along with the first DC vaccination.Randomization was stratified by CMV serology status and assignments weremade from a pool of consecutive sealed envelopes which had been preparedby the study statistician, using a random number generator. DCvaccinations began along with the first 5 day cycle (days 1-5) of TMZ(200 mg/m²). On day 21±2 of the TMZ cycle, both patient groups receivedan intradermal immunization every 2 weeks for a total of threeimmunizations with 2×10⁷ pp65-LAMP mRNA loaded mature DCs. Eachimmunization was divided equally to both inguinal regions. A totalvolume of 200 μL per side was delivered intradermally(SOP-JHS-HDC-CL-012 “Intradermal Administration of Dendritic CellsProcedure”). Details of the procedure were recorded on the appropriateform (FORM-JHS-HDC-CL-012). Injection was performed using a 1.5 inch 25gauge needle. Patients were monitored in the clinic for thirty minutesto one hour post-immunization for the development of any adverseeffects. The immunization procedures were supervised by a nurse orphysician that had completed an Advanced Cardiac Life Support (ACLS)course. A cardiac resuscitation cart was available in the immediatevicinity when performing these immunizations in the event of severeallergic reactions. The initial three vaccines were given each 2±1 weekapart.

After the third vaccine, patients underwent a 4-hour leukapheresis forimmunologic monitoring and DC generation. Subsequently, patients wereimmunized on day 21±2 of every cycle of TMZ which was delivered every5±1 weeks. Thus, in addition to the concurrent TMZ (75 mg/m²/d) givenwith radiation, a total of 6 addition cycles of TMZ (200 mg/m²/days 1-5)was given.

If the TMZ cycles were completed without progression, DC vaccinationscontinued every 5±1 weeks until clinical or radiographic progressionwithout any additional prescribed anti-tumor therapy. During that timeperiod, patients were monitored clinically with routine physical andneurologic examinations and MMSE testing at every visit and with acontrasted-enhanced CT or MRI every 8±4 weeks. Peripheral blood wasobtained at each visit as well immunologic monitoring.

A phase II randomized, prospective clinical trial was undertaken toassess the immunogenicity and efficacy of targeting the immunodominantCMV integument protein, pp65, in patients with newly-diagnosed GBM usingpp65-RNA transfected dendritic cells (DCs). After resection andradiation with concurrent TMZ (75 mg/m²/d), patients received subsequentmonthly cycles of TMZ (200 mg/m²) simultaneous with intradermalvaccinations and were randomized to receive an ALT (3×10⁷/Kg) prior tovaccination. Subjects received vaccinations until there was evidence oftumor progression.

The results are shown in FIG. 5. Twenty-one patients were consented.Five progressed during radiotherapy. There were no vaccine-related,reportable serious adverse events (SAEs). TMZ therapy, however, inducedGrade 3 lymphopenia (500 cells/mL) in 70% of patients after the firstTMZ cycle. After TMZ, immunosuppressive regulatory T cell (T_(Reg))(CD4⁺CD25⁺⁺CD45RO⁺CD127⁻FOXP3⁺) levels increased from 5.2% (3.3-7.5) to11.8% (6.9-13.8) (P=0.0004; paired t-test). One nearly complete responsewas observed. Median PFS was 12.5 months (CI₉₅: 10.0, ∞). Overallsurvival of patients with GBM treated with CMV pp65 RNA loaded DCvaccines is favorable. 6 PFS (100%; 13 of 13 pts), 12 month survival(92.3%; 12 of 13 pts), and 15 month survival (90%; 9 of 10 pts) comparefavorably to patients with similar diagnosis (see, e.g., Temodar® (63.7weeks) (Stupp et al., N Engl J Med., 352:987 (2005); and Gliadel™ (59.6weeks) (Westphal et al., Neuro Oncol., 5:79-88 (2003)). Overall mediansurvival is greater than 19.7 months. As shown in FIG. 6, a 33 year-oldfemale with GBM had nearly complete radiographic response to CMV pp65RNA-loaded DC vaccine combined with autologous lymphocyte transfer. MRIsshowed decrease in enhancing lesion, correction of midline shift, andresolution of neurologic symptoms. This patient remained alive and wellat 21 months post primary surgical resection of GBM.

Preliminary results of CMV-specific polyfunctional cellular and humoralimmune responses show that patients with GBM have CMV-specificimmunologic deficiencies (FIGS. 7-9).

Example 10 Antigen-Specific CD8+ and CD4+ T Cells can be Separated fromBulk Culture Responding to CMV pp65 Antigen-Pulsed DCs

To identify and isolate polyclonal populations of antigen-specific CD4+and CD8+ T cells, green fluorescent protein (GFP) RNA electroporationwas conducted on T cells stimulated with DCs transfected with mRNAencoding the full-length pp65 antigen of CMV. Dendritic cell generation,antigen loading with peptide and RNA, and maturation of dendritic cells:

All cellular materials used in these experiments were obtained fromnormal volunteers and patients with malignant glioma after informedconsent had been given, under the approval of the Duke UniversityInstitutional Review Board. Peripheral blood mononuclear cells (PBMCs)were obtained by leukapheresis and incubated for 1 hr in AIM-V medium(Invitrogen Life Technologies, Grand Island, N.Y.) at 37° C. to allowadherence to plastic and then cultured for 6 days in AIM-V mediumsupplemented with granulocyte-macrophage colony-stimulating factor (800units/ml) and IL-4 (500 units/ml). Immature dendritic cells wereharvested on day 6, washed, and resuspended in Opti-MEM (Invitrogen LifeTechnologies) at 2.5×10⁷/ml. The supernatant from DC culture was savedas conditioned medium for later use. Cells were electroporated in 2-mmcuvettes: 200 μl of DCs (5×10⁶ cells) at 300 V for 500 μsec, using asquare waveform generator (ECM 830 Electro Square Porator; BTX, adivision of Genetronics, San Diego, Calif.). DCs were electroporatedwith 2.5 μg of RNA per 10⁶ cells. Cells were transferred to 60-mm tissueculture Petri dishes containing a 1:1 combination of conditioneddendritic cell growth medium and fresh medium. Cells were maturedovernight with IL-1β (5 ng/ml), tumor necrosis factor-α (5 ng/ml), IL-6(150 ng/ml), and prostaglandin E₂ (1 μg/ml). IL-4, tumor necrosisfactor-α, IL-1β, and IL-6 were obtained from R&D Systems (Minneapolis,Minn.); granulocyte-macrophage colony-stimulating factor was from thepharmacy at Duke University Medical Center (Durham, N.C.) andprostaglandin E₂ was from Pharmacia (Erlangen, Germany). For peptidestimulation, mature dendritic cells were washed and resuspended in 1:1conditioned medium and fresh medium with a 10 μg/ml concentration of theHLA-A2-restricted immunogenic human cytomegalovirus pp65 peptideNLVPMVATV (pp65₄₉₅₋₅₀₃) (SEQ ID NO:109) (AnaSpec, San Jose, Calif.).

Pulsing of Dendritic Cells with pp65 Peptide or mRNA and Activation of TCells:

DCs were generated from HLA-A2⁺ normal volunteers and patients withmalignant glioma and pulsed with pp65 peptide (10 μg/ml) in AIM-Vmedium-2% human AB serum for 3 hr at 37° C. For loading with mRNAencoding pp65, DCs were washed and resuspended in Opti-MEM forelectroporation. Two micrograms of mRNA encoding full-length HCMV pp65per 1×10⁶ DCs was electroporated with the BTX ECM 830. DCs were washedonce with 45 ml of AIM-V medium and autologous responder lymphocyteswere added at a 1:10 ratio (DC:T cells). Volumes were adjusted to 2×10⁶cells/ml and cells were incubated at 37° C. in a humidified atmospherecontaining 5% CO2. After 3 days, an equal amount of AIM-V medium with 2%pooled human AB serum plus IL-2 (10 U/ml) was added and cells weretransferred to 24-well plates at a volume of 1 ml/well. Thereafter,every 2-3 days, cells were evaluated for growth and adjusted to 1×10⁶cells/ml. After 8 to 11 days of coculture with pp65-pulsed DCs, the Tcells were harvested and electroporated with mRNA encoding GFP.Alternatively, T cells were stimulated by polyclonal stimulation usingimmobilized anti-CD3 monoclonal antibodies (Ortho Biotech, Raritan,N.J.) (2 μg/ml in phosphate-buffered saline [PBS] overnight in a T-150flask). Cells were plated in AIM-V medium with 2% human AB serum (10⁶cells/ml) in CD3-coated plates and cultured for 3 to 5 days beforeharvesting.

Source of T Lymphocytes:

Nonadherent (NA) cells were generated from PBMCs from patients withmalignant gliomas and normal volunteers under Duke UniversityInstitutional Review Board approval after informed patient consent wasgiven. Mononuclear cells from peripheral blood were isolated byFicoll-Hypaque gradient separation (LSM; MP Biomedicals, Solon, Ohio).Electroporation of DC-pulsed pp65 peptide-stimulated T cells with GFP:

The mRNA encoding GFP was prepared from the PGEM4Z/GFP/A64 vector(kindly provided by E. Gilboa, Duke University Medical Center) aspreviously described (Zhao et al., Blood, 102:4137 (2003)). DCs pulsedwith pp65 peptide were used to stimulate autologous T cells as describedabove. The cells were kept in medium with 10 U/ml of IL-2 and incubatedfor 7 days at 37° C. For electroporation, 0.2 ml of the cell suspension(5×10⁶) was mixed with 10 μg of in vitro-transcribed GFP mRNA. Themixtures were electroporated in 0.4-cm cuvettes (EquiBio; PEQLABBiotechnologie, Erlangen, Germany) at 300 V and 150 Mf, using anelectroporation device. After electroporation, the cells were culturedin fresh AIM-V medium supplemented with 2% human AB serum and incubatedovernight. Twenty-four hours after electroporation, the cells wereharvested and stained for CD8 and HLA-A2 tetramer positivity andanalyzed by flow cytometry.

Expansion of Sorted T Cells:

Using the same stimulation and electroporation methods as describedabove, GFP-positive and negative T cells were sorted on a FACSVantage SEflow cytometer (BD Biosciences, San Jose, Calif.) 24 hr posttransfection. The cells were placed in AIM-V medium supplemented with100 U/ml of IL-2 to allow for expansion. Live cells were counted on days0, 7, 10, 14, and 17, using light microscopy and trypan blue dyeexclusion.

GFP Expression in Anti-CD3-Activated T Cells:

Stimulation of T cells with immobilized anti-CD3 antibody 48-96 hrbefore electroporation resulted in high levels of GFP expression in60-70% of T cells as analyzed by flow cytometry (FIG. 10A).

Selection of CMV Antigen-Specific CD8+ T Cells, Using RNAElectroporation of GFP:

The requirement for T cell activation for efficient RNA transfectioncould allow for the identification of antigen-specific T cells withinbulk stimulated PBMC cultures. To determine the efficiency of RNAelectroporation in antigen-specific T cells, PBMCs were stimulated for 7days with autologous DCs pulsed with an HLA-A2-restricted pp65 peptide.Day 7 stimulated cultures were electroporated with GFP RNA andexpression of GFP was examined in antigen-specific and nonspecific Tcells, using an HLA-A2 pp65 tetramer. Forward and side scatter gatingrevealed activated lymphocyte cells of blastlike morphology (FIG. 11,top left). GFP expression was shown in approximately 25% of all CD8+ Tcells (FIG. 11, top right), and tetramer analysis revealed thatexpression was restricted almost exclusively to pp65-specific T cells(FIG. 11, bottom right). Of T cells expressing GFP protein, 97% wereidentified as pp65 specific by tetramer staining. Furthermore, more than98% of all tetramer-positive CD8+ T cells expressed GFP protein,indicating that RNA transfection not only was restricted to antigenstimulated T cells (demonstrating exquisite specificity; 97.40%), butthat this process also efficiently identified all of the relevantresponder T cells (demonstrating high sensitivity for identifyingantigen-specific T cells; 89.77% sensitivity for identifying allrelevant cells). These results were confirmed in three repeatexperiments, with a mean specificity for transfection ofantigen-specific T cells of 93.10±5.87% and sensitivity of detecting alltetramer-positive T cells by GFP expression of 86.45±8.45%. GFPexpression in DC-stimulated T cells cultured in IL-2 (10 U/ml) was foundto persist for 5-7 days (FIG. 12A), indicating the capacity totransiently express proteins in rapidly expanding T cells.

Enrichment and Expansion of CMV Antigen-Specific CD8+ and CD4+ T Cells,Using RNA Electroporation of GFP:

To identify and isolate polyclonal populations of CMV antigen-specificCD4+ and CD8+ T cells, GFP RNA electroporation was conducted on T cellsstimulated with DCs transfected with mRNA encoding the full-length pp65antigen. Eleven days after in vitro stimulation with pp65 RNA pulsedDCs, PBMCs from HLA-A2-positive patients (n=3) were electroporated withGFP RNA and the kinetics of RNA expression were evaluated by flowcytometry (FIG. 12A). Gene expression was found to peak at 48 hr and todecline to baseline by day 7 posttransfection. Therefore, in repeatexperiments T cells were electroporated on day 11 after DC stimulationand 48 hr later GFP-positive (GFP+) and GFP-negative (GFP−) T cells wereseparated by flow cytometric cell sorting. Sorted cells were analyzed bytetramer analysis and cytokine flow cytometry for enrichment of CMVantigen-specific T cells. Equal numbers of GFP+ and GFP− cells wereplaced back into culture containing IL-2 (100 U/ml) and expansion wasevaluated on days 7, 10, 14, and 17 postsorting. Interestingly, onlyGFP+ T cells were capable of further expansion when cultured with IL-2,indicating that RNA electroporation can efficiently separate T cellscapable of further expansion in vitro from anergic or inactivated Tcells poststimulation with RNA-pulsed DCs (FIG. 3B). To determinewhether GFP expression was simply separating proliferating T cells fromanergic cells, GFP+ and GFP− sorted cells were also stimulated with apolyclonal expansion platform consisting of allogeneic feeder cells(PBMCs from an unmatched normal donor), anti-CD3, and high-dose IL-2(100 U/ml) in a rapid expansion protocol (REP) suitable for generatingclinical scale T cell expansions within 14 days (×1000-fold expansion).Using the REP protocol, both GFP− and GFP+ T cells were expandedefficiently, demonstrating that the capacity for RNA transfection doesnot segregate functional from nonfunctional T cells, but ratheridentifies T cells that have received sufficient activation during thecoculture with antigen pulsed dendritic cells to enter mitotic cellcycling.

One of the three patient samples examined demonstrated expansion of bothHLA-A2- and HLA-B7-restricted pp65-specific CD8+ T cells afterstimulation with RNA-pulsed DCs. After RNA electroporation and sorting,the GFP+ fraction contained 95% of the HLA-A2-restricted T cells and 98%of the HLA-B7-restricted T cells, representing a 20- to 50-foldenrichment of antigen-specific T cells within the GFP+ fraction, anddemonstrating efficient identification and separation of almost all ofthe antigen-specific CD8+ T cells (FIG. 12C). Similar results wereobtained with two other patients who demonstrated only HLA-A2-restrictedpp65 T cell reactivity. To determine the capacity to enrich for CMVantigen-specific CD4+ T cells, cytokine flow cytometry was performedwith unstimulated, Staphylococcus enterotoxin B (SEB)-stimulated, or CMVpp65 peptide mix-stimulated T cell fractions. The positive control(SEB-stimulated T cells) indicated that 13.69% of the CD4+ T cellssecreted IFN-γ in the GFP-negative fraction and 22.49% of the GFP+ CD4+T cells secreted IFN-γ, confirming that both fractions containedfunctional effector CD4+ T cells capable of responding to superantigen.However, IFN-γ secretion in response to the CMV peptide mix by CD4+ Tcells was restricted to the GFP+ fraction, with 14.15% of CD4+ T cellssecreting cytokine compared with 0.31% of CD4+ T cells in the GFP−fraction (FIG. 12D).

The results showed a 45-fold enrichment of antigen-specific CD4+ T cellswithin the GFP+ fraction after separation of T cells stimulated withpp65-pulsed DCs and demonstrated an effective method of identifyingantigen-specific CD8+ and CD4+ T cells within bulk PBMC cultures forenrichment. Furthermore, the capacity to identify and enrich polyclonalpopulations of antigen-specific CD8+ and CD4+ T cells provides a methodfor generating populations of T cells with broad antigen recognition andeffector function for use in adoptive immunotherapy.

Example 11 Antigen-Specific T Cells can be Modified to Migrate Toward aReceptor-Specific Chemokine In Vitro and In Vivo

Dendritic cell generation, antigen loading with peptide and RNA,maturation of dendritic cells, pulsing of dendritic cells with pp65peptide or mRNA, source of T lymphocytes, and activation of T cells wereperformed as described in Example 7 above except that CXCR2 mRNA wasused for electroporation of DC-pulsed pp65 peptide-stimulated T cells.CXCR2 mRNA was prepared from the pcDNA3.1+ vector (Missouri S&T cDNAResource Center, Missouri University of Science and Technology, Rolla,Mo.) and expressed in the Psp73-sph/A64 vector (also kindly provided byE. Gilboa) (Nair et al., Blood, 102:964 (2003)).

For cell migration assays, activated lymphocytes (5×10⁵ cells) wereplaced into the upper chamber of six-well filter chamber plates (BeckmanCoulter, Fullerton, Calif.) in triplicate. Medium containing no cytokineor various concentrations of IL-8, GRO-α, or UL146 was placed into thelower chamber and cells were incubated for 45 min to 1 hr at 37° C.Medium in the lower chamber was collected, and cells were centrifugedand resuspended in 50 ml of medium and counted by trypan dye exclusion.For assays of in vivo migration into the peritoneal cavity, unmodifiedor CXCR2 RNA-transfected T lymphocytes were differentially labeled withcarboxyfluorescein succinimidyl ester (CFSE; Invitrogen, Carlsbad,Calif.) at 1 μM (untransfected) and 10 μM (CXCR2 RNA transfected) for 5min in PBS, washed, and injected intravenously into recipient NOD/SCIDmice (Taconic, Hudson, N.Y.) at 10⁷ cells per mouse. One microgram ofchemokine (IL-8, UL146, or GRO-α) in 1 ml of PBS was injectedintraperitoneally every 8 hr for 24 hr after injection of T cells. Micewere sacrificed, lymphocytes were harvested by peritoneal lavage with 3ml of PBS, and cells were concentrated by centrifugation. Cells werestained with phycoerythrin (PE)-conjugated anti-human CD45 (BDBiosciences) and accumulation of untransfected (CFSE^(low)) and CXCR2RNA transfected (CFSE^(high)) cells was evaluated by flow cytometry. Therelative accumulation of T cell populations was determined by usingaccumulation in PBS-treated animals as a baseline for comparison ofresponse to chemokine injection. For in vivo migration assays into theCNS, 1 μg of UL146 in 5 μl of PBS was injected into the right parietallobe of mice under general anesthesia, using a stereotactic frame, and 5μl of PBS was injected into the left parietal lobe. CFSE (5 μM)-labeleduntransfected or CXCR2 RNA-transfected T cells were injected intorecipient mice at 10⁷ cells per mouse. Six hours later, mice weresacrificed and brains were harvested and dissected into right and leftparietal lobes. Single-cell digests were prepared by collagenasedigestion (1 mg/ml in RPMI) (Sigma-Aldrich, St. Louis, Mo.) of mincedtissue for 30 min at 37° C. Single-cell suspensions of left and rightparietal lobes were assessed for accumulation of infused lymphocytes byflow cytometric analysis of CFSE-labeled cells; 100,000 events werecollected and total numbers of infiltrating T cells were determined byflow cytometric counting of CFSE-positive cells.

Expression of CXCR2 mRNA in Antigen-Specific T Cells:

The capacity to modulate the migratory function of T cells by expressionof the chemokine receptor CXCR2, after CXCR2 RNA electroporation into Tcells stimulated with pp65-pulsed DCs was examined. CXCR2 expression wasmonitored in tetramer-positive versus tetramer-negative CD8+ T cells,using CXCR2-specific antibodies and flow cytometry. 10-15% of stimulatedCD8+ T cells expressed CXCR2 on their surface (FIG. 10B) at baseline andafter CXCR2 RNA transfection approximately 50% of CMV antigen-specific Tcells expressed CXCR2 on their surface (FIG. 13). Tetramer-negative Tcells, in contrast, did not exhibit CXCR2 expression above background(10.9%), indicating selective expression of the chemokine receptor inantigen-specific T cells after RNA electroporation.

Enhanced Chemotactic Function of CXCR2 RNA-Modified T Cells In Vitro:

The chemotactic function of unmodified, GFP RNA-transfected, and CXCR2RNA-transfected T cells in Transwell migration assays in response to theCXCR2-specific ligands IL-8, GRO-α, and UL146 was also examined (FIG.14A-C). CXCR2 RNA-transfected T cells exhibited a dose-responsiveenhanced chemotactic response to all three ligands compared withunmodified or GFP RNA-transfected T cells. The chemotactic responsetoward IL-8, secreted at high levels within a number of tumors, andUL146, a chemokine produced by human cytomegalovirus, and the onlyligand exclusive to CXCR2, were increased more than 300-500% inCXCR2-modified T cells.

Enhanced Migration of CXCR2 RNA-Modified T Cells In Vivo:

To determine whether transfection of CXCR2 RNA into activated T cellscould facilitate T cell migration in vivo, the migration of RNA-modifiedT cells into the peritoneal cavity and CNS of SCID mice that had beeninjected with CXCR2-specific chemokines was evaluated. Mice wereinjected intraperitoneally with chemokines (1 μg/ml) every 8 hr for 24hr post infusion of differentially CFSE-labeled untransfected or CXCR2RNA-transfected T cells. Mice were sacrificed and cells were harvestedby peritoneal lavage, and the accumulation of CXCR2-transfected anduntransfected T cells was analyzed by flow cytometry by gating on humanCD45+ cells and evaluating the relative accumulation of CFSE^(low) cells(untransfected) to CFSE^(high) cells (CXCR2 RNA transfected). Therelative accumulation of T cells in chemokine-injected mice was comparedwith that of PBS-injected mice and is shown in FIG. 15A. Results showedthe accumulation of CXCR2 RNA transfected cells in response to UL146 andIL-8. The greatest chemotactic response was to UL146, which paralleledfindings of in vitro studies. No significant accumulation was observedin response to GRO-α in this in vivo assay.

Whether CXCR2 RNA modification could lead to accumulation of T cellswithin the CNS in response to UL146 also was examined. Immunodeficientmice were administered a single injection of PBS in the left frontalparietal lobe, or of UL146 (1 μg) in the right frontal parietal lobe,under guidance of a stereotactic frame. CFSE-labeled untransfected orCXCR2 RNA-transfected T cells were injected intravenously and 6 hr postinjection, left and right cerebral hemispheres were harvested, singlecell digests were prepared, and detection of CFSE-labeled T cells wasevaluated by flow cytometry. As shown in FIG. 15B, CXCR2 RNA-modified Tcells exhibited enhanced infiltration of the UL146-injected hemispherescompared with PBS-injected hemispheres, whereas untransfected T cellsexhibited no significant difference in infiltration of eitherhemisphere. Preferential accumulation of CXCR2 RNA-modified T cells atlater time points was not observed, likely secondary to the transientgradient of chemokine established by the intracranial injection.

These results showed that CXCR2 RNA-modified T cells exhibit enhancedchemotactic function in vitro and in vivo and that RNA-basedmodification of T cells can be used to selectively identify and modifyantigen-specific T cells.

Thus, a gene transfer technology employing the electroporation ofmessenger RNA encoding genes that could be used to identify and separatetransfected from untransfected T cell populations, as well as modifytheir migratory function, was utilized. Using GFP as an example of amarker gene, CMV antigen-specific T cells were readily identified andseparated from cultures stimulated with pp65-pulsed DCs, and that RNAtransfection can be used to simultaneously isolate both antigen-specificCD4+ and CD8+ T cells. Nucleic acid-mediated transfection ofantigen-specific T cells has particular advantages in being able tosimultaneously enrich the purity and modify the function ofantigen-specific T cells by a single strategy, such as theelectroporation of chemokine receptor or other surface receptors ofinterest and isolation of cells on the basis of the expression of thetransfected receptor. The expression of genes encoding proteins that canmodulate the function of T cells, such as CXCR2, can be targetedpreferentially to those T cells with a specificity of interest, using anantigen specific stimulation platform followed by RNA electroporation,for example. Genes that enhance the migration of T cells to sites ofinflammation, viral infection, or tumor progression, such as chemokinereceptors, represent a class of proteins whose expression mightsignificantly enhance the efficacy of adoptively transferredlymphocytes. CXCR2 is normally expressed only in a small fraction of Tlymphocytes and mediates chemotaxis toward IL-8, GRO-α, and UL146, thelatter a CMV antigen-specific chemokine. IL-8 has been shown to beincreased in sites of inflammation as well as dramatically elevatedwithin gliomas and other tumors. GRO-α is increased in expression in anumber of malignancies including melanoma, gastrointestinal cancers, andmalignant gliomas. Its expression in gliomas correlates with tumorgrade, rendering T cells with chemotactic function toward GRO-α, auseful effector in tracking down invasive high-grade lesions. UL146 is achemokine synthesized by CMV with specificity for CXCR2. Thelocalization of CMV antigen-specific T cells expressing CXCR2 to sitesof viral infection through UL146-mediated chemoattraction can enhancethe efficacy of immunotherapy.

Additionally, these studies at least demonstrate that T cells can belocalized, for example to sites of therapeutic interest, using RNAsencoding chemokine receptors or other chemotactic receptors. This is ofparticular use in the targeting of brain tumors, as the CNS displayslimited normal trafficking of circulating immune cells. Among otherthings, this approach also can direct antigen-specific effector T cellsto sites of tumor growth, viral infection, or vaccine sites forpreferential expansion.

Example 12 Qualitative CMV Detection

To develop a qualitative CMV detection method, 29 different PCR primers(Table 1) spanning 10 different CMV genes were evaluated for detectingCMV DNA in an expanded cohort of blood samples from patients withnewly-diagnosed GBM (223 serial blood samples from 45 patients withnewly-diagnosed GBM).

Peripheral blood from normal volunteers (median age 42 for healthyvolunteers (n=11) and age 46 for surgical control patients (n=6)),patients with newly-diagnosed GBM (median age 52.5 (n=45)), and patientsundergoing allogeneic bone marrow transplantation (median age (n=5)) wascollected in accordance with the Duke University Institutional ReviewBoard. Whole blood was aliquoted into cryotubes and snap frozen inliquid nitrogen and stored at −130° C. until DNA extraction. Plasma andserum was collected by centrifugation of heparinized or non-heparinizedblood respectfully at 2000 g for 20 min and snap frozen in cryotubes inliquid nitrogen and stored at −130° C. until DNA extraction. Tumors werecollected during surgery after informed consent from patients withnewly-diagnosed GBM, minced and snap frozen in liquid nitrogen.Specimens were stored at minus 130° C. until DNA extraction. DNA wasextracted from 10 mg of minced tissue using TissueDirect™ Multiplex PCRSystem (GenScript Corporation, Piscataway, N.J.).

DNA was extracted from GBM tumor specimens and human blood or plasma orserum with TissueDirect™ Multiplex PCR System (GenScript Corporation,Piscataway, N.J.). For positive control samples, whole blood, serum, orplasma from seronegative volunteers was spiked with known concentrationsof CMV Quantitated Viral DNA AD169 Strain (Advanced Biotechnologies(Cat#08-925-000), Columbia, Md.). Lysis solution from the TissueDirect™Multiplex kit was made by mixing solution TD-A with solution TD-B at 1:9ratio. For DNA extraction, various ratios of Lysis solution to samplevolume were evaluated and 50 μL of Lysis solution TD-A/B was added to 10μL of blood (serum or plasma or tumor tissue) per sample, mixed well bypipetting up and down or tapping/rotating the tube, and spun briefly.The samples were incubated at 95° C. for 15-30 minutes being sure tokeep caps tight to prevent loss due to evaporation until sample wasuniformly lysed by Lysis solution. 50 μL of solution TD-C was added toeach sample, mixed well and spun at high speed on microcentrifuge forone minute and supernatants collected. 11 μL of 5M NaAC or 3M NH₄AC(1:10 volume of lysed sample volume) and 220 μL of 100% Alcohol (EtOH, 2volumes of sample volume) were added to the supernatant forprecipitating DNA. The DNA pellets then centrifuged at full speed(14,000 rpm) in a microcentrifuge for at least 10 minutes and washedtwice with 70% EtOH and reconstituted with 10 μL of Nuclease-Free wateror TE buffer. All DNA extractions were performed in a separatelaboratory from the molecular biology lab in which PCR reactions werecarried out. 2-5 μL of each sample (equivalent to the DNA extracted from2-5 μL of whole blood, plasma, or serum) was added to PCR reactions forCMV detection.

Qualitative PCR detection was run using TissueDirect™ Multiplex PCRSystem (GenScript Corporation, Piscataway, N.J.) according tomanufacturer's instructions and visualization by gel electrophoresis.Twenty-nine different primers spanning 10 CMV genes were selected frompublished literature or designed on Vector NTI Advance 9 software(Invitrogen, Carlsbad, Calif.) and synthesized by Integrated DNATechnologies (IDT, Coralville, Iowa). DNA extracted from 2-5 μL ofsample (whole blood, serum, or plasma) or tumor tissue was carefullyadded to each 50 μL of PCR reaction. A sample from a CMV seronegativedonor and DNase-Free water was used in parallel as a PCR negativecontrol. PCR was run on iCycler (Bio-Rad, Hercules, Calif.) at 94° C.for 15 min., 40 cycles of 94° C. for 40 sec., annealing temperature for1 min. and 72° C. for 1 min, and ended with extension at 72° C. for 10min. PCR products were visualized by electropheresis on CriterionPrecast 10% of polyacrymide gel (Bio-Rad (Cat#345-0053), Hercules,Calif.) in 1×TBE (Bio-Rad, Hercules, Calif.) and stained with SYBR GoldNucleic Acid Gel Stain (Invitrogen (Cat#S11494), Carlsbad, Calif.). Forconfirmation of CMV DNA detection, some amplified DNA bands wereisolated from gels with QIAEXII (Qiagen, Valencia, Calif.) followingmanufacturer's protocol and sequenced at Duke University ComprehensiveCancer Center DNA Sequencing Facility. Sequence identity was analyzedusing BLAST of NCBI database and alignment analysis of specimensconducted using Vector NTI Advance 10 (Invitrogen, Carlsbad, Calif.).

As shown in Table 1, the detection rate of CMV viremia in peripheralblood samples of patients with GBM ranged from 12% to 73.5% depending onthe primers utilized in the PCR assay. Primers within the gB gene (e.g.,B-i1i2) were found to be the most sensitive for detection of CMV in theperipheral blood of patients with GBM. No viral DNA was detected incontrol lanes (water only) and the peripheral blood of normal volunteerswere negative for detection of viral DNA upon several repeat assays (0out of 17 samples including 11 CMV seropositive volunteers; p=8.4×10⁻¹⁰;Fisher's exact test). The detection rate of CMV in the expanded cohortof 223 blood samples (164 positive out of 223 samples) did not differsignificantly from initial findings of 16 of 20 positive blood samples(p=0.603; Fisher's exact test) (data not shown).

In order to evaluate the effect of amplicon size on CMV detection rate,the overall detection rate of CMV in the blood of patients with GBM forall primers was compared based on amplicon size alone by Spearman andPearson correlation coefficient analysis. Primers designed to giveamplicons of 200 bp or less were also found to have a lower threshold ofdetection of CMV and more reliable amplification (Table 1). For example,primers gB E1E2 (amplicon size 268), gBi1i2 (amplicon size 144), andgBi3i4 (amplicon size 122) all span the same region of DNA within the gBgene and differ only by the distance between the primer sets. Theobservation that smaller amplicons gave a higher rate of detection wasmade with primers in other CMV genes as well. An inverse correlationbetween amplicon size and detection rate was shown for the 29 primersinvestigated independently of the gene evaluated. The PearsonCorrelation Coefficient (r=−0.37622) and Spearman CorrelationCoefficients (r=−0.41523 were both significant (p=0.0443 and 0.0251respectively). These results indicate the profound effects of primerselection and amplicon size in the detection rate of CMV DNA inperipheral blood of patients with GBM.

To investigate the impact of PCR amplification directly from sampleswithout DNA purification, BloodReady™ Kit (GenScript Corporation,Piscataway, N.J.) was compared to TissueDirect™ Kit (GenScriptCorporation, Piscataway, N.J.) for the direct amplification of DNA fromwhole blood and tumor tissue. PCR amplification can be performeddirectly from cell lysates after DNA extraction or after purification ofDNA from other organic molecules present within in cellular lysates.

Human tissues was kept on ice immediately after surgery and minced into10 mg size within less than half hour, then snap frozen in liquidnitrogen and stored at −137° C. until to use. Patient or normal bloodwas aliquoted 10 μl per tube and snap frozen in liquid nitrogen andstored at −137° C. until to use.

Genomic DNA Preparation: Used TissueDirect™ DNA Preparation andMultiplex PCR Kit (Cat#L00195; GenScript, Piscataway, N.J.): 1. ThawedBuffers TD-A, TD-B and TD-C at room temperature and placed on ice afterthawing; 2. Mixed 5 μl of TD-A and 45 μl of TD-B (1:9 ratio) for eachsample and spun to yield 50 μA of lyses solution (TD-AIB) per sample; 3.Added 50 μl of lysis solution TD-AB to each sample 10 mg of tissue or 10μl of blood (serum or plasma) and mixed well by pipetting up and down ortapping/rotating the tube; 4. Incubated the samples at 65° C. for atleast 10 minutes until complete lyses of sample occurred; 5. Removedtubes from incubation, added 50 μl of TD-C to each tube, and mixed well;6. Spun samples at ˜14,000 rpm for at least 1 minute; 7. Stored thesupernatant (genomic DNA extract) at 4° C. or proceeded with PCRamplification; 8. Used 1.5 μl of the genomic DNA extract in the 20 μl ofPCR per sample.

PCR Amplification:

A. Qualitative measurement: PCR amplification was performed withTissueDirect™ DNA Preparation and Multiplex PCR Kit (Cat#L00195;GenScript, Piscataway, N.J.) or HotMaster™ Taq PCR system (Brinkmann,Westbury, N.Y.):

Pre-Mix Solution is shown in Table 4.

TABLE 4 PreMix GenScript Multiplex PCR System 1X PCR H₂O  8.2 μl SensePrimer (lug/ul) 0.15 μl Antisense Primer (1 ug/ul) 0.15 μl GenScript 2XPCR premix   10 μl Pre-Mix Volume 18.5 μl Genomic DNA to be added  1.5μl Total Volume   20 μl Annealing Temp. 60° C.

Added 1.5 μl of genomic DNA solution to 18.5 μl of pre-mix shown inTable 4 to give 20 μl of PCR solution per tube. Placed PCR tubes intoBio-Rad iCycler (Bio-Rad, Milpitas, Calif.) and cycled as follows: 1cycle of 94° C. for 5 minutes (Denaturation); and 40 cycles of: 94° C.for 40 seconds (Denaturation)/60° C. for 40 seconds (Annealing)/72° C.for 40 seconds (Extension)/1 cycle of 72° C. for 10 minutes (Extension).

B. Quantitative Measurement: Real-Time PCR amplification with iQ SYBRGreen SuperMix (2× Mix for Real-Time, Bio-Rad Cat. #170-8880) or iQSuperMix (2× SuperMix for Real-Time, Bio-Rad Cat. #170-8860):

Pre-Mix Solution is shown in Table 5.

TABLE 5 PreMix GenScript PCR 1X PCR H₂O 16.7 μl iQ SYBR Green SuperMixor   20 μl iQ SuperMix with Gene Specific Probe Sense Primer (lug/u1)0.15 μl Antisense Primer (lug/u1) 0.15 μl Pre-Mix Volume   37 μlGenoinic DNA to be added   5 μl Total Volume   40 μl Annealing Temp. 60°C. PCR product bp

Added 3 μl of genomic DNA to 37 μl of pre-mix shown in Table 5 to yield40 μl of PCR solution. Placed the PCR solution into Byroad iCycler andcycled as follows: 1 cycle of 95° C. for 3 minutes (Denaturation); and40 cycles of: 94° C. for 30 seconds (Denaturation)/60° C. for 30 seconds(Annealing)/72° C. for 30 seconds (Extension)/1 cycle of 72° C. for 10minutes (Extension).

Following amplification, Real-Time PCR products were analyzed withstandard curve and the data normalized with housekeeping genes.

PCR Product Detection: Criterion™ Precast 10% Gel (26 Well, 15 μl/well)in TBE (Cat. #345-0053; BioRad, Hercules, Calif.) was used essentiallyas follows: Added 1×TBE buffer to electrophoresis chamber up to the fillline. Mixed 2.5 μl of DNA Loading Buffer with 10 μl of PCR product persample to yield 12.5 μl aliquots. Added 1.5 μl of EZ DNA MolecularWeight Standard (100 bp MW, BioRad Cat#170-8352, 0.05 μg/μl, 500 μl)into first well of gel. Added 12.5 μl aliquots of PCR products to wells2-26. The gel was run at 120 voltages for 2 hrs at room temperature(RT). Electrophoresis was terminated and transfer each gel into 25 ml ofStaining buffer (SYBR Gold Nucleic Acid Gel Stain, Invitrogen/MolecularProbe Cat#511494) in 1×TBE. Gels were stained for 15 minutes at RT.Photographed gel picture using different exposure times to record andanalyze the PCR products. Each right size of PCR product was then cutout from the gel and the DNA was extracted using QIAEX II (Qiagen,Valencia, Calif.). The extracted DNA was then sequenced.

As shown in FIG. 16, TissueDirect™ Kit was reliable for providing cleanand distinct amplifiable bands from both whole blood and tumor samples.Also PCR results using the TissueDirect™ Kit on small sample volumes(10-20 μL) were compared to two commercial DNA Purification Kits:DNeasy® (Qiagen, Valencia, Calif.) and Gentra® PureGene® (Qiagen,Valencia, Calif.) which require larger sample volume (0.2 ml to 1.0 mlof whole blood, serum, or plasma) for DNA extraction. No difference inthe lower limits of viral DNA detection was found when using theTissueDirect™ Kit compared to purifying DNA from larger sample volumes(not shown), indicating that the simpler and faster direct DNAextraction method is equally effective and may be preferable,particularly when sample volume is limiting. The melt curve graph ofHCMV gp64 Real-Time PCR from GBM patient sera is shown in FIG. 17 andthe PCR amplification cycle graph of HCMV gp64 Real-Time PCR from GBMpatient sera is shown in FIG. 18.

Moreover, the ratio of sample to tissue Lysis buffer was compared usinga sample:buffer ratio of 1:5, 1:10, and 1:20 and evaluated by PCRdetection of CMV DNA using the gB primer set (B-i1i2; Table 1) on 34known positive samples. As shown in Table 6, a sample to buffer ratio ofone to five gave detection with a strong, clear band of appropriate size(122 bp), while other ratios either gave reduced intensity of bandsignal or resulted in detection of non-specific bands.

TABLE 6 Comparison of extraction buffers and buffer:sample ratio indetection of CMV DNA. Buffer Ratio (Sample/ TissueDirect TD-A/BBloodReady BR-A Buffer) 2:1 1:1 1:2 1:5 1:10 1:20 1:5 1:10 1:20 Blood12/34 17/34 34/34 34/34 34/34 34/34  7/34 16/34 34/34 Plasma 17/34 21/3434/34 34/34 34/34 34/34 12/34 20/34 34/34 Serum 20/34 24/34 34/34 34/3434/34 34/34 14/34 23/34 34/34

In all cases, the predominant band corresponded to the gB gene product.Moreover, SYBR Gold Nucleic Acid Gel Stain (Invitrogen, Carlsbad,Calif.) gave an enhanced visualization of viral DNA bands compared toethidium bromide staining (not shown). A ratio of 1:5 was chosen for allsubsequent analysis.

There was no loss of viral DNA during genomic DNA extraction fromsamples within the single step protocol described herein, andspecifically this is very important to detect low level of viral DNA inthe samples thereby saving precious patient samples. The GenScriptTissueDirect™ Multiplex PCR System was employed for all blood, serum andtissues to make sure that the viral DNA is completely released into thelyses buffer (TD-A/B). Thus, we were able to obtain no viral DNA lossand limit inhibitors to PCR. Moreover, PCR primers were designed to makesmaller amplicons (less than 200 bp) with high gene specificity and nocross-binding to human genome for more efficient and sensitive PCRamplification within the high human genome content and no purificationto remove inhibitors to PCR. The PCR condition amplified gene specificproducts at very low level of target DNA. The detection sensitivity wasincreased at least 10 fold compared to conventional agarose gel stainedwith Ethidium Bromide after polyacrylamide gel electrophoresis. SYBRGold staining and digital photograph were all optimized together,thereby avoiding transferring separated PCR products from agarose gelsto membrane for gene specific probe hybridization to detect viral DNA orNEST PCR.

Example 13 Quantitative CMV Detection

To provide a quantitative PCR assay for measuring CMV DNA levelsspecifically in patient samples, 15 gene-specific primer and probecombinations (Table 2) were evaluated for quantifying CMV DNA copynumber in whole blood spiked with CMV whole genomic DNA as a positivecontrol standard using real-time PCR.

Real-Time PCR detection was run in TaqMan® Universal PCR Master Mix(Applied Biosystems, Foster City, Calif.) by following manufacturer'sinstruction. Fifteen different gene-specific primer & probe setsspanning 5 CMV genes were chosen from published literature or designedwith Primer 3.0 software (Applied Biosystems, Foster City, Calif.) inthe lab and synthesized by Integrated DNA Technologies (IDT, Coralville,Iowa). DNA from 2-5 μl of sample (whole blood, serum, or plasma) wasadded to each 50 μL PCR reaction. PCR was run on 7900HT (AppliedBiosystems, Foster City, Calif.) by following manufacturer's standardprotocol at specified annealing temperature. The copy number wasanalyzed with SDS 2.3 software (Applied Biosystems, Foster City, Calif.)based on standard curve from CMV Quantitated Viral DNA.

As shown in Table 2, the gB primer/probe set gB21/22/23 displayed thelowest threshold for detection of CMV standards using limiting dilutionsof genomic CMV DNA, the highest frequency of CMV DNA detection inperipheral blood specimens from patients with GBM, and the earliestlogarithmic amplification cycle (Ct value) of the probe and primer setsevaluated.

There were significantly different Ct values amongst the fifteenprimer/probe combinations evaluated, with Ct values ranging from 31.5 to38.1. This indicates over a 100-fold difference in the sensitivity ofvarious primer/probes in detection of identical concentrations of CMVDNA standards in the blood. The differences in sensitivity betweenprimer/probe sets resulted in false negatives using less sensitive probecombinations at limiting levels of CMV standards and also falsenegatives in blood samples from patients with GBM (Table 2).

Example 14 Detection of Viral Reactivation in Patients UndergoingAllogeneic Bone Marrow Transplant

To further examine the qualitative and/or quantitative PCR methodsdescribed in Examples 12 and 13, the detection of CMV viremia inpatients being monitored serially for viral reactivation afterundergoing allogeneic bone marrow transplantation (aBMT) was evaluated.

Thirty serum samples obtained serially from 5 patients after aBMT wereevaluated using the qualitative and quantitative PCR assays of theinvention and also in the Duke Clinical Microbiology Laboratory usingthe CMV UL54 analyte-specific reagent (ASR) test (Roche Diagnostics,Indianapolis, Ind.). The testing laboratory was blinded to the resultsof the ASR test, and patient samples were evaluated as to whether theywere positive or negative by the qualitative laboratory PCR assay aftergel electropheresis analysis and viral load in copy number per ml ofblood was determined using the quantitative laboratory PCR assay.

Detection of CMV DNA by ASR test conducted by the Clinical MicrobiologyLaboratory at Duke University Medical Center and the qualitative andquantitative PCR tests are shown in Table 7.

TABLE 7 Comparison of laboratory PCR tests to diagnostic PCR assay RocheLab Quantitative Lab Qualitative Date UL54 PCR PCR # Patient collectedASR gB gB 1 AB 20-Jun 1121 424 Pos 2 AB 27-Jun 2998 254 Pos 3 AB 4-Jul3150 237 Pos 4 AW 10-Jan 0 187 Pos 5 AW 7-Feb 0 45 Pos 6 BH 30-May 0 0Neg 7 BH 6-Jun 0 0 Neg 8 BH 13-Jun 0 0 Neg 9 BH 20-Jun 0 375 Pos 10 BH27-Jun 0 0 Neg 11 BH 4-Jul 0 0 Neg 12 BH 11-Jul 0 0 Neg 13 BH 18-Jul 01781 Pos 14 BH 25-Jul 0 196 Pos 15 BH 1-Aug 1561 262 Pos 16 FD 27-Jun 00 Neg 17 FD 11-Jul 0 0 Neg 18 FD 18-Jul 0 0 Neg 19 FD 25-Jul 6335 649Pos 20 FD 1-Aug 5705 1640 Pos 21 JAG 10-May 0 0 Neg 22 JAG 18-Jul 0 0Pos 23 JAG 25-Jul 0 0 Neg 24 JAG 1-Aug 0 305 Neg 25 OptiQuant 5,000 nt206 Pos 26 OptiQuant 500,000 nt 7739 Pos 27 TB 27-Jun 0 0 Pos 28 TB6-Jul 0 0 Pos 29 TB 25-Jul 0 0 Neg 30 TB 1-Aug 0 0 Neg

Positive results were confirmed by isolation of DNA bands after gelelectropheresis and DNA sequencing, indicating no false positives by ourPCR test. After analysis, results from the clinical diagnosticevaluation of these patients by the ASR test were unblinded. The PCRtest was positive in all cases of CMV viremia deemed positive by theRoche CMV UL54 ASR qPCR test conducted by the Clinical MicrobiologyLaboratory indicating no false negatives by the PCR test of theinvention. However, the PCR tests of the present invention detectedviral DNA in several patient samples prior to detection of viralreactivation by the clinical diagnostic tests (Table 7). In some cases,viremia was detected six weeks prior to the first positive result by theclinical diagnostic assay. These results were confirmed as truepositives by DNA sequencing of amplified bands and identified as CMVusing NCBI DNA database.

These results indicate that the laboratory PCR method of the inventionis accurate for detection of CMV DNA and capable of detecting viralreactivation in patients undergoing aBMT that is below the threshold ofdetection of the Roche UL54 ASP assay. These results highlight theutility of this assay as an easier, faster, and more sensitive detectionassay detection of CMV DNA and warrant further study to validate the useof this assay for prophylactic monitoring of patients at risk for CMVdisease as well as investigating the association of CMV with diseasessuch as malignancy and atherosclerosis where detection has beencontroversial.

Example 15 Sequencing of gB (UL55) DNA from GBM Tumor SpecimensIdentifies Viral Genotype

The identity of viral strains associated with GBM tumors wasinvestigated by PCR amplification and DNA sequencing of a strainvariable region of the gB (UL55) gene. CMV gB DNA from twenty-twosurgically resected GBM specimens was amplified using the gBi1i2 primerset, DNA analyzed by gel electropheresis, and amplified bands excisedand subjected to DNA sequencing. DNA sequences were searched using theNCBI DNA database for sequence identity and all DNA matched to CMV gB.The strain identity and gB family was determined by the examination ofthe NCBI database for DNA sequence identity. The strain(s) with highestdegree of homology are listed in Table 8.

TABLE 8 Identity & Strains of HCMV gBi1i2 PCR Sequences for 22 GBMPatients Identity % Identity % Identity % Identity % Compared ComparedCompared Compared gB Patient to 1T (88 bp) to 17T (85 bp) to 20T (99 bp)to 21T (99 bp) Strains Subtypes  1T 100 99 91 90 Merlin N1 gB1 (97%)  2T91 92 86 87 Merlin N1 gB1 (86%)  3T 92 94 90 91 Towne gB1 (90%)  4T 9496 94 89 AD169 N12 gB2 (92%)  5T 95 99 94 92 Merlin Towne N1 gB1 (94%) 6T 93 95 91 88 AD169 N12 gB2 (93%)  7T 95 96 90 93 Merlin Towne N1 gB1(92%)  8T 94 95 88 90 Merlin N1 gB1 (97%)  9T 93 95 91 86 AD169 N12 gB2(96%) 10T 96 98 90 93 Towne gB1 (96%) 11T 96 100 92 89 Merlin Towne N1gB1 (93%) 12T 98 96 89 91 Merlin N1 gB1 (95%) 13T 88 90 83 84 Merlin N1gB1 (92%) 14T 100 99 90 89 Merlin N1 gB1 (96%) 15T 96 100 92 93 MerlinN1 gB1 (97%) 16T 94 96 97 92 AD169 N12 gB2 (95%) 17T 99 100 95 94 TownegB1 (97%) 18T 92 94 91 89 AD169 N12 gB2 (91%) 19T 94 98 94 93 Towne gB1(95%) 20T 91 95 100 89 AD169 N12 gB2 (94%) 21T 90 94 89 100 Towne gB1(93%) 22T 88 90 86 94 Merlin N1 gB1 (93%)

Sequence alignment of viral DNA from the GBM specimens revealed uniqueviral isolates from the GBM specimens with DNA sequence variation up to17% between clinical isolates (FIG. 19; and Table 8). Comparison ofamplified viral DNA gB sequence to gB gene sequences within the NCBIdatabase was also conducted and the highest degree of homology to gBgenotypes are listed in Table 8. In cases where identical degree ofhomology to more than one gB genotype was revealed, the viral strainidentity was used to determine the gB genotype. Several isolates had thesame degree of homology with more than one strain of CMV and were listedaccordingly. Strains from GBM specimens were found to be homologous withMerlin (gB isotype 1), N1 (gB isotype 1), Towne (gB isotype 1), AD169(gB isotype 2), and N12 (gB isotype 2) strains of CMV, indicating thatdistinct viral isolates were detected in tumor specimens, and also thatGBM tumors are permissive for infection by the gB1 and gB2 familygenotypes of CMV virus. The distribution of gB genotypes observed wasgB1 16/22 (72.7%); gB2 6/22 (27.3%).

These results at least demonstrate that GBM specimens are morefrequently infected by CMV strains of the gB1 genotype. Several patienttumor samples were re-analyzed months apart beginning with DNAextraction from cryopreserved whole blood or tumor specimens, PCRamplification, and DNA sequencing of gB amplicons, and identical viralsequences were obtained demonstrating the fidelity of viralidentification in these samples.

The consistent detection of an association between CMV and variousdisease states has been controversial, with conflicting findings byvarious laboratories reporting on the presence of viral DNA and proteinswithin pathologic lesions. The above examples describe PCR-based assaysand the strain identity of 22 clinical strains of CMV detecting insurgically resected GBM specimens. The development of the CMV PCR testsfor reliable detection of CMV in accordance with the present inventionalso can, among other things, facilitate studies into the role CMV playsin glioma development and progression. GBM tumors were found to beinfected by strains matching the gB1 and gB2 genotypes with apredominance of gB1 type viruses. The gB1 genotype is the most prevalentviral family found in clinical CMV infections in the U.S. but the lackof any detection with the highest degree of homology matching clinicalstrains of the gB3 or gB4 genotypes suggests that there may be arestricted tropism for CMV infection of malignant gliomas.

Detection of viral DNA within tumor specimens is not a trivial issueeven in tumors where the association has been well established such asin the case of human papilloma virus associated cervical cancers, anddetection can often be at or below the limits of sensitivity by standardPCR assays. As shown above, several parameters were determined thatenhanced the detection of low levels of viral DNA in the blood andtumors of patient with newly-diagnosed GBM.

Primers spanning the same region of DNA within the gB gene and differingonly by the distance between the primer sets demonstrated an ampliconsize dependent frequency of detection of viral DNA. gBi1i2 and gB-i3i4,which have the smallest amplicon sizes within this region also gave thehighest detection rates, indicating that false negative results can bedue to inefficient amplification of larger DNA fragments and not theabsence of viral DNA within samples when viral DNA may be limiting.Analysis of 29 independently derived CMV primer sets spanning 11different genes demonstrated an inverse correlation between ampliconsize and detection rate of CMV DNA. These results suggest that falsenegative PCR detection by others of limiting amounts of viral DNA can bedue to ineffective extraction and amplification conditions and cannotnecessarily be deemed to be conclusive. Moreover, extraction andimplication conditions can account for false negative results in viralDNA detection.

The majority of the amplified products were confirmed by DNA sequencingand in all cases CMV specific DNA was amplified. And, in order torelease viral DNA more efficiently from samples, blood was snap frozenin liquid nitrogen, then incubated at higher temperature (95° C.) thanthe suggested temperature of 65° C. by the TissueDirect™ kit afteraddition of lysis buffer. Also incorporated was an EtOH precipitationstep after DNA extraction in order to concentrate DNA and eliminate PCRinhibitors that are often present in the crude sample extracts. Forqualitative PCR, a combination of polyacrylamide gel and SYBR Goldstaining provided increased PCR detection sensitivity of at least 10fold compared to conventional agarose gel stained with ethidium bromide.This increased sensitivity allowed detection without use of moreintensive protocols, such as nested PCR or gene specific probehybridization of blotted DNA. This assay has the added advantage ofbeing able to utilize small volumes of specimen (10-20 μL) allowingrepeated analysis of samples and preservation of patient samples thatmay be limiting. Furthermore, the evaluation in patients undergoing bonemarrow transplantation demonstrated that the assay is capable of earlierdetection of viral reactivation in immunosuppressed patients thancurrently available diagnostic PCR tests.

It has been reported that CMV has an association and possible pathogenicrole in a number of disease processes including cancer, atherosclerosis,and inflammatory bowel disease. The detection of CMV in association withthese diseases, however, has varied considerably amongst investigator'slaboratories. The relatively simple and enhanced amplification detectionmethod that can utilize small volumes of blood and tissue specimens canallow for more reliable determination of the association of CMV withsuch disease processes.

What is claimed is:
 1. A method for eliciting in a subject an immuneresponse to a cancer cell that expresses a CMV antigen, the methodcomprising: selecting a subject in need of treatment related to thepresence of the cancer cell; detecting the expression of the CMV antigenby the cancer cell in the subject, and administering to the subject apharmaceutically acceptable composition comprising at least one CMVantigen, wherein the pharmaceutically acceptable composition, whenadministered to the subject, elicits an immune response to the cancercell.
 2. The method of claim 1, wherein the at least one CMV antigencorrespond to a polypeptide, or immunogenic fragment thereof, selectedfrom the group consisting of: capsid polypeptides, tegumentpolypeptides, and envelop polypeptides.
 3. The method of claim 1,wherein the at least one CMV antigen correspond to a polypeptide, or animmunogenic fragment thereof, selected from the group consisting of:phosphoprotein unique lon g83 (ppUL83; a/k/a pp65), glycoprotein UL55(gpUL55; a/k/a gB), UL123 immediate early I (IE1) protein, UL122 IE2protein, UL111A (a/k/a mtrII), US28, ppUL32, ppUL65, ppUL80a, ppUL82,ppUL98a, ppUL99, pgUL4, (a/k/a gp48), gpUL16, gpUL18, (a/k/a MHC),gpUL75 (a/k/a gH), gpUL100, gpUL110 (a/k/a gM), gpUL115 (a/k/a gL),pUL46, pUL48, pUL56, pUL86 (a/k/a MCP), glycoprotein unique short 10(gpUS10), gpUS11, glycoprotein complex II (gcII), gp65, and pg93.
 4. Themethod of claim 1, wherein the pharmaceutically acceptable compositionfurther comprises at least one adjuvant, or a nucleic acid encoding theadjuvant.
 5. The method of claim 4, wherein the adjuvant is a cytokineselected from the group consisting of: GM-CSF, G-CSF, IL-2, IL-4, IL-7,IL-12, IL-5, IL-21, TNF-α, and M-CSF.
 6. The method of claim 1, whereinthe at least one CMV antigen comprises an amino acid sequencecorresponding to SEQ ID NO:109.
 7. The method of claim 1, wherein thecomposition further comprises a glycoprotein UL55 (gpUL55; a/k/a gB) oran immunogenic fragment thereof.
 8. The method of claim 1, wherein thecomposition further comprises a UL123 immediate early 1 (IE1) protein oran immunogenic fragment thereof.